Clinical trial NCT01222767
Conditions: Ewing's Sarcoma Primitive Neuroectodermal Tumor (PNET) Askin's Tumor of the Chest Wall Extraosseous Ewing's Sarcoma (EOE)
Keywords: EFT PNET EOE
Sponsor: PharmaMar
Start date: 2010-12-01; Completion date: 2012-04-01
Enrollment: 17 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is a phase II Multicenter, Open-label, Clinical and Pharmacokinetic Study of Zalypsis® (PM00104) in Patients with Unresectable Locally Advanced and/or Metastatic Ewing Family of Tumors (EFT) Progressing After at Least One Prior Line of Chemotherapy to determine the antitumor activity of Zalypsis.

Detailed description:

Arms: arm 1: None
Interventions: intervention 1: Zalypsis
Intervention descriptions: intervention 1: Zalypsis is provided as a lyophilized powder for concentrate for solution for infusion in a strength of 2.5 mg/vial.

Protocol documents:
Prot_000.pdf: 
 
 
 
SPONSOR 
 
Pharma Mar, S.A. 
Avda de los Reyes, 1 
 
Polígono Industrial “La Mina” 
28770 Colmenar Viejo (Madrid), Spain 
Phone: + 34 918 466 000  
Fax:  
+ 34 918 466 003 
 
 
Pharma Mar USA, Inc. 
One Liberty Plaza, 23rd floor, suite 2335 
New York, NY 10006, USA 
 
Phone: +1 212 201 6770 
Fax: +1 212 201 6771 
 
 
 
 
CLINICAL TRIAL PROTOCOL 
 
Phase II Multicenter, Open-label, Clinical and Pharmacokinetic Study of Zalypsis® 
(PM00104) in Patients with Unresectable Locally Advanced and/or Metastatic Ewing 
Family of Tumors (EFT) Progressing After at Least One Prior Line of Chemotherapy 
 
 
INVESTIGATIONAL MEDICINAL PRODUCTS: PM00104 (Zalypsis®) 
Protocol No.: PM104-B-003-10 
EudraCT: 2010-022221-15 
NCT Code: 01222767 
 
 
 
FINAL version 1.0: 30 July 2010 
 


SPONSOR(S)
Pharma Mar S.A., Sociedad Unipersonal
Pharma Mar USA, Inc.
Avda de los Reyes, 1
One Liberty Plaza, 23rd floor,
Polígono Industrial “La Mina”
suite 2335
28770 Colmenar Viejo (Madrid) Spain
New York, NY 10006, USA
Phone:+ 34 91 846 6000 
Phone:+1 212 201 6770
Fax:
+ 34 91 846 6003
Fax:
+1 212 201 6771
CLINICAL TRIAL PROTOCOL
PM104-B-003-10
Phase II Multicenter, Open-label, Clinical and Pharmacokinetic Study of Zalypsis®
(PM00104) in Patients with Unresectable Locally Advanced and/or Metastatic Ewing 
Family of Tumors (EFT) Progressing After at Least One Prior Line of Chemotherapy
INVESTIGATIONAL MEDICINAL PRODUCT/STUDY DRUG: Zalypsis® (PM00104)
Protocol Code: PM104-B-003-10
EudraCT:
2010-022221-15
FINAL version 1.0: 
30 July 2010
This study will be conducted in compliance with the protocol, GCP and applicable 
regulatory requirements.
Confidentiality Statement
The Information and data included in this protocol contain trade secrets and privileged or 
confidential information which is the property of the Sponsor(s). No person is authorized to 
make it public without written permission of the Sponsor(s). These restrictions on disclosure 
will apply equally to all future information supplied to you which is indicated as privileged or 
confidential.  This material may be disclosed to and used by your staff and associates as it may 
be necessary to conduct the clinical study.


STUDY 
CONTACTS
NAME AND ADDRESS
PHONE AND FAX 
NUMBER, AND E-MAIL 
ADDRESS
Pharma Mar S.A., 
Sociedad Unipersonal 
(hereafter referred as 
PharmaMar)
Avda. de los Reyes, 1
Polígono Industrial “La Mina”
28770 Colmenar Viejo (Madrid), 
Spain
Phone: + 34 91 846 6000
Fax:
+ 34 91 846 6003
PharmaMar USA, Inc.
One Liberty Plaza, 23rd floor,
suite 2335
New York, NY 10006, USA
Phone:
+1 212 201 6770
Fax:
+1 212 201 6771
Responsible Physician
Pilar Lardelli
Clinical Oncology
Clinical Development, 
PharmaMar 
Phone:
+ 34 91 846 6087
Fax:
+ 34 91 823 4504
E-mail: plardelli@pharmamar.com
Project Manager 
Manuel Luque
Clinical Operations 
Clinical Development, 
PharmaMar 
Phone:
+ 34 91 823 4548
Fax:
+ 34 91 846 6003
E-mail: mluque@pharmamar.com
Isabel Ibarra Cabo
Pharmacovigilance. 
Clinical Development.
PharmaMar
Phone: + 34 91 846 6081
Fax:
+ 34 91 846 6004
E-mail: iibarra@pharmamar.com
Pharmacovigilance 
Contact
Outside office hours:
Elena Roy
Pharmacovigilance.
Clinical Development, 
PharmaMar 
Phone: + 34 91 823 4749
E-mail: eroy@pharmamar.com
Clinical Pharmacology 
Contact
Carlos Fernandez Teruel
Clinical Pharmacology
Clinical Development
PharmaMar
Phone: + 34 91 846 6156
Fax:
+ 34 91 823 4504
E-mail:cfteruel@pharmamar.com
Central Laboratory 
for Pharmacokinetic 
Samples
Alan Gibbs
Icon Development Solutions.
Manchester Bioanalytical 
Laboratory. 
Parkway One, Parkway Business 
Centre, 300 Princess Road, 
Manchester, M14 7QU, United 
Kingdom.
Phone: + 44 161 232 2819
Fax:
+ 44 161 227 6565
E-mail: alan.gibbs@iconplc.com
Pharmacogenomics 
Contact
Juan Carlos Tercero
Scientific Development. 
PharmaMar 
Phone: + 34 91 846 6023
Fax:
+ 34 91 846 6001
E-mail: 
jctercero@pharmamar.com
Pharmacogenomic 
RNA analysis
Miquel Tarón Roca
Head of Molecular Biology 
Laboratory Pangaea Biotech S.A.
C/ Sabino Arana, 5
Consultas Externas. Planta -1 
(-1.7)
08028 Barcelona, Spain
Phone: + 34 93 497 8925
Fax: 
+ 34 93 497 8950


Tissue sample 
Processing and 
Pathology and 
Immunohistochemistry 
Analyses
Enrique de Álava
Hospital Universitario de 
Salamanca
Pº De San Vicente, 58
37007 Salamanca, Spain
Phone: + 34 923 294820
Fax: 
+ 34 923 294795
E-mail: edealava@usal.es


PRINCIPAL INVESTIGATORS
A complete list of Investigators, including the Principal Investigators, will be available 
as a separate document.


TABLE OF CONTENTS
PRINCIPAL INVESTIGATORS..............................................................................................................4
TABLE OF CONTENTS...........................................................................................................................5
SYNOPSIS ..................................................................................................................................................7
SCHEDULE OF ASSESSMENTS AND PROCEDURES....................................................................18
1.
INTRODUCTION .......................................................................................................................22
1.1.
Ewing’s Family of Tumors (EFT).......................................................................................22
1.1.1.
Therapeutic approach for EFT...................................................................................................... 22
1.2.
Zalypsis® (PM00104) .........................................................................................................23
1.2.1.
Chemical Structure and Formulation............................................................................................ 23
1.2.2.
Mechanism of Action ................................................................................................................... 23
1.2.3.
Preclinical Data ............................................................................................................................ 24
1.2.4.
Metabolism................................................................................................................................... 26
1.2.5.
Clinical Data................................................................................................................................. 26
1.2.5.1.
Clinical Trial PM104-A-004-05......................................................................................... 28
1.2.5.2.
Pharmacokinetic Data........................................................................................................ 30
1.2.6.
Pharmacogenomic Data................................................................................................................ 30
1.2.6.1.
Rationale for the Pharmacogenomic Substudy Associated to PM104-B-003-10............... 31
1.3.
Rationale for this Phase II Exploratory Trial of Zalypsis® in EFT ....................................33
2.
STUDY OBJECTIVES................................................................................................................35
2.1.
Primary Objective...............................................................................................................35
2.2.
Secondary Objectives .........................................................................................................35
3.
OVERALL STUDY DESIGN......................................................................................................35
4.
SELECTION OF PATIENTS ......................................................................................................36
4.1.
Inclusion Criteria ...............................................................................................................36
4.2.
Exclusion Criteria...............................................................................................................37
4.3.
Patients for the Pharmacogenomic Substudy .....................................................................38
4.3.1.
Inclusion Criteria.......................................................................................................................... 38
4.3.2.
Exclusion Criteria......................................................................................................................... 38
5.
PLAN OF THE STUDY...............................................................................................................38
5.1.
Duration of Study (Whole Population)...............................................................................38
5.2.
Duration of Study and Treatment (Per Patient) .................................................................38
5.2.1.
Discontinuations........................................................................................................................... 39
5.2.1.1.
Treatment Discontinuation................................................................................................. 39
5.2.1.2.
Reasons for Treatment Discontinuation............................................................................. 39
5.2.1.3.
Study Discontinuation........................................................................................................ 40
5.2.2.
Protocol Deviations ...................................................................................................................... 40
5.3.
Replacement of Patients .....................................................................................................41
5.4.
Study Termination (Clinical Cutoff) ...................................................................................41
5.5.
Pre-treatment Evaluation ...................................................................................................41
5.6.
Patient Registration............................................................................................................43
5.7.
Evaluations During Treatment ...........................................................................................43
5.8.
Evaluation at End of Treatment..........................................................................................44
5.9.
Follow-up after End-of-treatment Visit ..............................................................................45
6.
PHARMACOKINETICS.............................................................................................................45
6.1.
Sample Collection, Storage, and Shipping of the PK Samples ...........................................45
7.
TREATMENT .............................................................................................................................47
7.1.
Description of Treatment....................................................................................................47
7.2.
Administration of Study Medication ...................................................................................47
7.2.1.
Formulation.................................................................................................................................. 47
7.2.2.
Dose Schedule.............................................................................................................................. 47
7.2.3.
Criteria for Treatment Continuation ............................................................................................. 47
7.2.4.
Dose Reduction ............................................................................................................................ 48
7.3.
Concomitant Medication ....................................................................................................49
7.3.1.
Prophylactic Antiemetic Treatment.............................................................................................. 49
7.3.2.
Concomitant Medication Permitted.............................................................................................. 49
7.3.3.
Concomitant Medication Prohibited............................................................................................. 49
7.4.
Packaging and Labeling.....................................................................................................50


7.4.1.
Zalypsis® ...................................................................................................................................... 50
7.5.
Drug Accountability ...........................................................................................................50
7.6.
Treatment Compliance .......................................................................................................50
8.
STUDY EVALUATIONS ...........................................................................................................50
8.1.
Efficacy...............................................................................................................................50
8.2.
Safety ..................................................................................................................................51
8.3.
Central cardiological review..............................................................................................51
8.4.
Pharmacodynamic Substudy...............................................................................................51
8.5.
Pharmacogenomic Substudy...............................................................................................52
9.
ADVERSE EVENTS REPORTING............................................................................................53
9.1.
Definitions ..........................................................................................................................53
9.1.1.
Adverse Event .............................................................................................................................. 53
9.1.2.
Serious Adverse Event ................................................................................................................. 53
9.1.2.1.
Death.................................................................................................................................. 54
9.1.2.2.
Life-threatening Event ....................................................................................................... 54
9.1.2.3.
Hospitalization or Prolongation of Hospitalization............................................................ 54
9.1.3.
Unlisted/Unexpected Adverse Event............................................................................................ 54
9.1.4.
Assessment of Causal Relationship to the Study Drug/IMP......................................................... 55
9.1.5.
Adverse Events Related to Study Drug (IMP).............................................................................. 55
9.1.6.
Expedited Reporting..................................................................................................................... 55
9.2.
Procedures..........................................................................................................................55
9.2.1.
Reporting Adverse Events............................................................................................................ 55
9.2.2.
Reporting Serious Adverse Events ............................................................................................... 56
9.2.3.
Reporting Pregnancy Cases Occurring During the Clinical Trial................................................. 56
9.2.4.
Periodic Safety Review of Clinical Data ...................................................................................... 57
10.
STATISTICAL METHODS....................................................................................................58
10.1.
Sample Size.........................................................................................................................58
10.2.
Endpoints............................................................................................................................58
10.2.1.
Primary Endpoints................................................................................................................... 58
10.2.2.
Secondary Endpoints............................................................................................................... 58
10.3.
Statistical Analysis..............................................................................................................59
10.3.1.
Efficacy Analysis .................................................................................................................... 59
10.3.2.
Safety Analysis ....................................................................................................................... 60
10.3.3.
Pharmacokinetic Analysis....................................................................................................... 60
10.3.4.
Pharmacodynamic and Pharmacogenomic Analyses .............................................................. 60
11.
ADMINISTRATIVE SECTION .............................................................................................60
11.1.
Ethics..................................................................................................................................60
11.2.
Monitoring, Auditing and Inspecting..................................................................................61
11.3.
Patient Informed Consent Form.........................................................................................61
11.4.
Confidentiality / Identification of Patients..........................................................................62
11.5.
Case Report Forms.............................................................................................................62
11.6.
Insurance............................................................................................................................62
11.7.
Retention of Records...........................................................................................................62
11.8.
Use of Information and Publications..................................................................................63
12.
REFERENCES........................................................................................................................64
APPENDIX 1. ECOG PERFORMANCE STATUS ASSESSMENT SCALE ....................................67
APPENDIX 2. PERCENT OF BONE MARROW IRRADIATED......................................................68
APPENDIX 3. RESPONSE EVALUATION CRITERIA IN SOLID TUMORS (RECIST) V. 1.1 ..69
APPENDIX 4. COCKCROFT AND GAULT’S FORMULA...............................................................89
APPENDIX 5. DECLARATION OF HELSINKI .................................................................................90


SYNOPSIS
TITLE
Phase II Multicenter, Open-label, Clinical and Pharmacokinetic 
Study of Zalypsis® (PM00104)  in Patients with Unresectable 
Locally Advanced and/or Metastatic Ewing Family of Tumors 
(EFT) Progressing After at Least One Prior Line of 
Chemotherapy.
PROTOCOL CODE
PM104-B-003-10
INVESTIGATORS / 
TRIAL LOCATION
A minimum of six centers in Europe and the USA are expected 
to participate.
A full list of investigators will be available as a separate 
document.
STUDY OBJECTIVES
Primary

To 
determine 
the 
antitumor 
activity 
of 
Zalypsis®
administered as a 1-hour intravenous (i.v.) infusion on Day 1, 8 
and 15 every four weeks (d1, d8 and d15, q4wk) to patients with 
advanced and/or metastatic EFT.
    Secondary

To determine time-to-event efficacy parameters.

To characterize the safety profile and tolerability of 
Zalypsis® in patients with unresectable advanced and/or 
metastatic EFT.

To characterize the pharmacokinetics (PK) of Zalypsis®
when administered as a single-agent to patients with EFT.

To determine the pharmacodynamic profile by measuring the 
effect of Zalypsis® on the number of Ewing’s sarcoma 
circulating tumor cells (CTCs) at different times of treatment and 
its correlation with the clinical outcome.

To 
determine 
the 
pharmacogenomic 
(PGx) 
profile. 
Hypothesis-generating exploratory PGx analyses will be 
conducted to correlate the molecular parameters found in the 
tumor and blood samples of the patients with the clinical results 
achieved with Zalypsis®.
STUDY DESIGN 
Multicenter, open label, phase II clinical trial with single-agent 
Zalypsis® given as a 1-hour i.v. infusion on d1, d8 and d15, 
q4wk, to patients with advanced and/or metastatic EFT who 
failed to standard chemotherapy.
The primary endpoint of the study is the overall response rate 
(ORR), defined as the percentage of patients with objective 
response (OR), either complete response (CR) or partial response 
(PR), as defined by the Response Evaluation Criteria in Solid 
Tumors (RECIST) v. 1.1.
The study will consist of two stages. If in the first stage there are 
no responders after testing the drug on 12 evaluable patients, the 
trial will be terminated. If the trial goes on to the second stage, 


17 additional evaluable patients will be accrued. A total of 29 
evaluable patients will be studied. If the total number of 
responders is ≤ 2, the drug will be considered as not interesting 
in the setting of patients treated for this disease with this 
schedule.
Treatment will be administered in the absence of disease 
progression and/or unacceptable toxicity. In case of obtaining a 
CR, two additional cycles will be administered and then the 
treatment will be stopped.
STUDY POPULATION
Patients with EFT progressing after standard treatment with 
systemic chemotherapy are eligible for this trial.
To be included in the study, patients have to fulfill all inclusion 
criteria and none of the exclusion criteria.
INCLUSION CRITERIA 1. Voluntary written informed consent, obtained from the 
patient or his/her representative before the beginning of any 
specific study procedures. 
2. Age ≥ 16 years.
3. Histologically or cytologically confirmed EFT, with recurrent 
disease.
4. Documented failure to at least one prior chemotherapy 
regimen for their disease.
5. Radiographic documentation of disease progression at study 
entry.
6. Eastern Cooperative Oncology Group (ECOG) performance 
status (PS) score ≤ 2.
7. Life expectancy ≥ 3 months. 
8. Complete recovery from the effects of drug-related adverse 
events (AEs) derived from previous treatments, excluding 
alopecia and grade 1 peripheral neuropathy, according to the 
National Cancer Institute Common Terminology Criteria for 
Adverse Events (NCI-CTCAE) v. 4.0.
9. At least one measurable lesion (“target lesion” according to 
the RECIST v.1.1), located in a non-irradiated area and 
adequately measured less than four weeks before study entry. 
Tumors within a previously irradiated field will be designated 
as "non-target" lesions unless progression is clearly 
documented or biopsy proven.
10.Absolute neutrophil count (ANC) ≥ 1.5 x 109/l; platelet count 
≥ 100 x 109/l, and hemoglobin ≥ 9 g/dl.
11.Adequate renal function: calculated creatinine clearance 
(using Cockcroft and Gault‘s formula) ≥ 30 ml/min.
12.Adequate hepatic function:
a) Total bilirubin  1.5 x upper limit or normality (ULN), 
unless due to Gilbert’s syndrome.
b) Alanine 
aminotransferase 
(ALT), 
aspartate 
aminotransferase (AST) ≤ 3 x ULN (≤ 5 x ULN in case 


of hepatic metastases), and alkaline phosphatase (AP) ≤ 
2.5 x ULN (≤ 5 x ULN in case of extensive bone 
involvement).
c) Albumin ≥ 25 g/l.
13.Left ventricular ejection fraction (LVEF) within normal limits 
(LVEF of at least 50%).
14.Women of childbearing potential must have a negative serum 
pregnancy test before study entry. Both women and men must 
agree to use a medically acceptable method of contraception 
throughout the treatment period and for three months after 
discontinuation of treatment. Acceptable methods of 
contraception include complete abstinence, intrauterine 
device (IUD), oral contraceptive, subdermal implant and 
double barrier (condom with a contraceptive sponge or 
contraceptive suppository).
Pharmacodynamic and 
pharmacogenomic 
substudy inclusion 
criteria

All patients included in trial PM104-B-003-10 will be 
eligible.

Only those patients who have themselves or their 
representatives voluntarily signed the Informed Consent Form 
for the PGx study will participate.
EXCLUSION 
CRITERIA
1. Prior therapy with Zalypsis®.
2. Pregnant or lactating women or women of childbearing 
potential not using an appropriate contraceptive method. 
3. Less than three weeks from prior radiation therapy, biological 
therapy or chemotherapy.
4. Less than six weeks from prior nitrosourea, mitomycin C, 
high-dose chemotherapy or radiotherapy involving the whole 
pelvis or over 50% of the spine, provided that acute effects of 
radiation treatment have resolved. Hormonal therapy and 
palliative radiation therapy (i.e., for control of pain from bone 
metastases) must be discontinued before study entry. 
5. Patients with a prior invasive malignancy (except non-
melanoma skin cancer and in situ cervix carcinoma) who 
have had any evidence of disease within the last five years or 
whose prior malignancy treatment contraindicates the current 
protocol therapy.
6. Evidence of progressive or symptomatic central nervous 
system (CNS) metastases or leptomeningeal metastases.
7. Other diseases or serious conditions:
a)
Increased cardiac risk, as defined by:

Unstable angina or myocardial infarction within 12 
months before inclusion in the study.

New York Heart Association (NYHA) grade II or 
greater congestive heart failure. 

Symptomatic 
arrhythmia 
or 
any 
arrhythmia 
requiring ongoing treatment. 



Abnormal electrocardiogram (ECG), i.e., patients 
with the following are excluded: QT prolongation -
QTc > 480 msec; signs of cardiac enlargement or 
hypertrophy; bundle branch block; partial blocks; 
signs of ischemia or necrosis, and Wolff Parkinson 
White patterns.

History or presence of valvular heart disease.

Uncontrolled arterial hypertension despite optimal 
medical therapy.

Previous mediastinal radiotherapy.

Previous treatment with doxorubicin at cumulative 
doses exceeding 400 mg/m2.
b)
History of significant neurological or psychiatric 
disorders.
c)
Active infection requiring systemic treatment.
d)
Significant 
non-neoplastic 
liver 
disease 
(e.g., 
cirrhosis).
e)
Known hepatitis B virus (HBV) or hepatitis C virus 
(HCV) infection.
f)
Immunocompromised patients, including those known 
to be infected with the human immunodeficiency virus 
(HIV).
g)
Uncontrolled (i.e., requiring relevant changes in 
medication within the last month or hospital admission 
within the last three months) endocrine diseases (e.g., 
diabetes mellitus, hypo- or hyperthyroidism, adrenal 
disorder).
8. Any other major illness that, in the Investigator’s judgment, 
will substantially increase the risk associated with the 
patient’s participation in the study. The Investigator should 
feel free to consult the Study Coordinator or the Sponsor(s) in 
case of uncertainty in this regard.
9. Limitation of the patient’s ability to comply with the 
treatment or to follow-up at a participating center. Patients 
enrolled into this trial must be treated and followed at a 
participating center.
10.Treatment with any investigational product within 30 days 
prior to inclusion in the study.
11.Known hypersensitivity to any component of Zalypsis®.
Pharmacodynamic and 
pharmacogenomic 
substudy exclusion 
criteria

Patients who do not consent to participate in this substudy (by 
themselves or through their representatives). Refusal to 
participate in the PGx substudy will not affect participation in 
the main trial PM104-B-003-10.
No. of patients
A total of 29 evaluable patients are expected to participate in this 
trial.


No. of sites
A minimum of six centers are expected to participate.
STUDY DRUG
Formulation
Zalypsis®
Zalypsis® is provided as a lyophilized powder for concentrate for 
solution for infusion in strength of 2.5 mg/vial.
Route of administration
Intravenous, as a 1-hour infusion by a central catheter.
Administered dose
Zalypsis® will be administered at a dose of 2 mg/m2.
Treatment schedule
A treatment cycle consists of the drug administration on Day 1, 8 
and 15 (study evaluations should be completed during each cycle 
prior to subsequent Zalypsis® infusion). Treatment cycles will be 
repeated every four weeks.
Prophylactic medication
Antiemetic prophylaxis: patients will receive prophylactic 
treatment 
for 
emesis 
for 
adult 
patients 
consisting 
of 
dexamethasone 8 mg i.v. and 5-HT3 antagonists (ondansetron 8 
mg i.v., or granisetron 1 mg i.v., or tropisetron 5 mg i.v.) before 
the infusion of Zalypsis®, according to the American Society for 
Clinical Oncology (ASCO) guidelines for drugs with moderate 
emetic risk.
If necessary, one or both of the following may also apply:

Adding 10 mg of metoclopramide orally every eight 
hours.

Extending the duration of treatment with 5-HT3 
antagonists and/or dexamethasone.
Secondary prophylaxis with colony-stimulating factors such as 
granulocyte 
or 
granulocyte-macrophage 
colony-stimulating 
factors (G-CSF or GM-CSF), or with erythropoietin is allowed 
according to the ASCO guidelines.
Criteria for continuation 
of treatment and for re-
treatment
In order to be re-treated on Day 8 and Day 15 of the first cycle 
and on subsequent infusions, the patients will have to fulfill the 
following criteria: 
a)
Platelet count  75 x 109/l, hemoglobin  9 g/dl and 
ANC  1.0 x 109/l.
b)
AP ≤ 2.5 x ULN (≤ 5 x ULN in case of extensive bone 
involvement).
c)
ALT, AST ≤ 3 x ULN (≤ 5 x ULN in case of hepatic 
metastases).
d)
Renal function: patients with calculated creatinine 
clearance (using Cockcroft and Gault's formula)
 30 ml/min.
e)
Total bilirubin  1.5 x upper limit or normality (ULN), 
unless due to Gilbert’s syndrome.
f)
Albumin ≥ 2.5 g/dl.


g)
Other non-hematological toxicities grade  1 (grade 2 
in case of asthenia). 
If these criteria are not met on Day 8, 15 or on subsequent
infusions, study drug administration should be skipped and 
criteria reevaluated weekly. Treatment administration will be 
resumed upon recovery of these parameters, according to these 
same criteria.
If a patient has to skip two doses (at Day 8 and Day 15) or has a 
delay > 2 weeks from the theorical day of re-treatment, the 
patient should discontinue from treatment. In the event of 
obvious clinical benefit, the patient will remain on treatment only 
after having discussed and agreed upon the case with the 
Sponsor(s), and upon recovery of all parameters according to the 
aforementioned criteria.
Dose reduction
Zalypsis® dose reductions will take place based on the worst 
treatment-related 
toxicity 
found 
since 
the 
last 
dose 
administration. The criteria for dose reduction are:

Grade 4 neutropenia that lasts > 5 days.

Febrile neutropenia.

Grade 4 thrombocytopenia.

Grade ≥ 3 transaminase increase that lasts > 7 days.

Any other grade 3-4 adverse non-hematologic toxicity 
except nausea, vomiting and diarrhea (unless grade 3 or 
4 nausea, vomiting and diarrhea persists despite use of 
adequate medication).
If the patient requires dose reduction, the new Zalypsis® dose 
level will be one of those described in the table below:
Dose level
Zalypsis® (mg/m2)
1
2.0
-1
1.6
-2
1.3
No more than two dose reductions per patient (from 2 mg/m2 to 
1.6 mg/m2 and then to 1.3 mg/m2) will be allowed during the 
study. Patients requiring more than two dose reductions should 
discontinue the treatment, except in the event of obvious clinical 
benefit, in which case the patient could be allowed to remain on 
treatment after having discussed and agreed upon the case with 
the Sponsor(s).
No dose escalations will be allowed in this study.
EFFICACY 
EVALUATIONS
To be evaluable for efficacy:

patients must have received at least four of the six infusions 
in the first two cycles (e.g., two infusions in each cycle, or 
three infusions in Cycle 1 and one infusion in Cycle 2), AND



patients must have at least one disease measurement 
recorded not less than six weeks after treatment onset. 
In addition, any eligible patient who receives at least two of the 
three infusions in one treatment cycle and experience disease 
progression or dies due to progressive disease (PD) prior to 
response evaluation will be considered evaluable for the main 
endpoint (ORR) and will be categorized as an “early 
progression”.
Patients withdrawn due to toxicity without any tumor assessment 
after the start of study treatment will be considered as “treatment 
failures” and will not be replaced.
Patients withdrawn due to significant clinical deterioration of 
unknown reason, or due to hypersensitivity reactions or unrelated 
AEs, and patients who refuse to continue on study for any reason 
without any tumor assessment after the start of study treatment 
will be considered not evaluable for efficacy and will be 
replaced.
Assessment of efficacy will be done using the RECIST v. 1.1 and 
will be essentially based on a set of measurable lesions identified 
at baseline as target lesions and followed until disease 
progression. A disease evaluation will be performed at baseline 
and every other cycle (± 1 week) until evidence of PD. The same 
procedure will be used to evaluate each identified lesion both at 
baseline and throughout the treatment period.
In case of detection of an OR, either a CR or a PR, a 
confirmation assessment has to be performed after a minimum of 
four weeks from the first documentation of the response.
Additionally, 
detection 
of 
the 
presence of 
CTCs 
by 
retrotranscriptase polymerase chain reaction (RT-PCR) analysis 
of EWS-FLI1 and EWS-ERG translocations in the blood of 
patients will be performed.
SAFETY 
EVALUATIONS
Patients will be evaluable for safety if they have received at least 
one total or partial infusion of Zalypsis®. 
Safety will be evaluated using clinical examinations, which will 
comprise vital signs analysis, clinical assessment of AEs, 
changes 
in 
laboratory 
parameters 
(hematological 
and 
biochemical, including liver and cardiac function tests) and any 
other analyses that may be considered necessary.
All AEs will be classified according to the NCI-CTCAE v.4.0 
and will be coded using the Medical Dictionary for Regulatory 
Activities (MedDRA) v. 11.0.
PHARMACOKINETIC 
EVALUATIONS
The PK of plasma Zalypsis® will be evaluated during the first 
two infusions of the first cycle with a limited sampling schedule 
of eight samples. 
PK parameters will be calculated using population methods, after 
pooling data from this study with data obtained during phase I 
studies.


The sampling schedule during each infusion will be as follows: 
Sample number
Optimal time point
Adequate time frame
1
Pre SOI
Pre SOI
2
5 min before EOI
5-1 min before EOI
3
30 min after EOI
30 min-1 h after EOI
4
2 h after EOI
1.5-3 h after EOI
5
6 h after EOI
6-8 h after EOI
6
24 h after EOI
20-28 h after EOI
7
48 h after EOI
40-72 h after EOI
8*
168 h after EOI
120-168 h after EOI
*Sample number 8 has to be taken BEFORE the start of the next infusion.
SOI, start of infusion; EOI, end of infusion.
PHARMACODYNAMIC 
AND 
PHARMACOGENOMIC 
SUBSTUDY 
EVALUATIONS
The aim of this investigation is to identify and validate molecular 
markers whose expression may be associated with the clinical 
outcome of patients treated with Zalypsis®. These molecular 
markers might allow the identification of those patients who will 
benefit from the treatment with Zalypsis®, thus improving health 
care by an individualized medicine. 
The following analyses will be done on tumor and blood samples 
from consenting patients treated with Zalypsis®: 
Quantitation of mRNA expression in paraffin-embedded 
tumor tissue by quantitative real-time retrotranscriptase
polymerase chain reaction (qRT-PCR) of genes identified 
during in vitro studies as potential biomarkers of response 
to Zalypsis®. These genes will be selected from PDGFRa 
and PARP and other genes related to the mechanism of 
action (MoA) of Zalypsis®.
Quantitation of protein expression by immunohistochemistry 
in tissue microarrays constructed from the patient’s 
paraffin-embedded tumor tissue blocks. The proteins to be 
determined include PDGFRa and PARP, and other proteins 
related to the MoA of Zalypsis®.

Fluorescence 
in 
situ 
hybridization 
(FISH) 
and 
retrotranscriptase polymerase chain reaction (RT-PCR) 
analysis of EWS-FLI1 and EWS-ERG translocations and 
fusion protein variants in paraffin-embedded tissue.

Detection of the presence of CTCs by retrotranscriptase 
polymerase chain reaction (RT-PCR) analysis of EWS-
FLI1 and EWS-ERG translocations in the blood of patients.
STATISTICAL 
METHODS
Primary Endpoint:
Overall response rate (ORR), defined as the percentage of 
patients with confirmed OR, either CR or PR, according to 
the RECIST v 1.1.
Secondary Endpoints:
Duration of response (DOR), defined as the time between the 
date when the response criteria (PR or CR, whichever is first 
reached) are fulfilled and the first date when disease 
progression, recurrence or death is objectively documented 


(taking the smallest measurements documented since the 
treatment started as reference for PD).
Progression-free survival (PFS), defined as the time from the 
first day of study treatment to the day of negative assessment 
(progression or death) or last tumor evaluation. PFS at 3 and 
PFS at 6 months are defined as the Kaplan-Meier estimates 
of PFS at these time points.
Overall survival (OS), defined as the time from the first day 
of treatment to the date of death (or the last day when the 
patient is known to be alive). Survival will be followed every 
three months until death, or until the date of study 
termination, whichever occurs first. OS at 12 months is 
defined as the Kaplan-Meier estimates of OS at this 
timepoint.
Safety profile.
Pharmacokinetic profile.
Pharmacodynamic profile
Pharmacogenomic profile.
Sample Size Considerations: 
The optimum two-stage design to test the null hypothesis that the 
ORR is ≤ 3% versus the alternative that ORR ≥ 20% was 
selected. After testing the drug on 12 patients in the first stage, 
the trial will be terminated if there is not any responder. If the 
trial goes on to the second stage, a total of 29 patients will be 
studied, 12 from the first stage and 17 from the second stage. If 
the total number of responding patients is ≤ 2, the drug will be 
considered not interesting in the setting of patients treated with 
this disease and with this schedule. This design has an expected 
sample size of 17.20 and a probability of early termination of 
0.69. If the drug is actually not effective, there is a 0.05 
probability of concluding that it is (type I error). If the drug is 
actually effective, there is a 0.1 probability of concluding that it 
is not (type II error).
Methods of Analysis:
Binomial estimates with exact 95% confidence intervals will be 
calculated for the analysis of the main endpoint (ORR).
Time-to-event endpoints (DOR, PFS and OS, PFS rates at 3, 6 
months and OS rates at 12 months) will be analyzed according to 
the Kaplan-Meier method. 
Baseline characteristics, AEs, serious adverse events (SAEs), 
laboratory evaluations, deaths and the reason for study 
discontinuations will be analyzed. Continuous variables will be 
tabulated and presented with summary statistics (i.e., mean, 
standard deviation, median and range). Categorical variables will 
be summarized in frequency tables by means of counts and 
percentages.
DURATION OF STUDY 
PERIOD (per patient)
Patients will be evaluated at scheduled visits in up to three study 
periods:


Pre-treatment: from signature of informed consent to the 
first infusion of Zalypsis®.
Treatment: from the first infusion of Zalypsis® to the last 
administration of the study drug plus 30 days, or onset of 
subsequent therapy, or death, whichever occurs first. An 
end-of-treatment visit will be performed within 30 days after 
last dose administration.
Follow-up: all patients will be followed after the end of 
treatment:

Patients 
who 
discontinue 
treatment 
without 
progression will be followed every three months until 
disease progression, other antitumor therapy, or death, 
or until the date of study termination, whichever 
occurs first. 

After disease progression or after other antitumor 
therapy, patients will be followed every three months 
until death, or until the date of study termination, 
whichever occurs first.
Study termination (clinical cutoff) will vary depending on the 
development of the trial. Planned study termination will occur:

Depending on the development/extension of the trial: 
o
Three months after the last treatment visit of the 
last evaluable patient if the study ends at the first 
stage.
o
Six months after the last treatment visit of the 
last evaluable patient, if the study proceeds to 
the second stage.

Or 12 months after the last patient included in the study, 
if neither of the above have occurred.
Patients will be considered to be on-study from the signature of 
the informed consent to the end of follow-up period. Patients will 
be considered to be on-treatment for the duration of their 
treatment and in the first 30 days following the date of the last 
study drug administration, or until the onset of subsequent 
therapy or death, whichever occurs first. End of treatment is 
defined as the day of the last study drug dose administration plus 
30 days, or the onset date of subsequent therapy or the date of 
death, whichever occurs first.
Patients will receive Zalypsis® while it is considered to be in 
their best interest. Specifically, treatment will continue until:
Disease progression.
Unacceptable toxicity (including any toxicity leading to the 
need for a third dose reduction or severe hypersensitivity 
reactions, except in the event of obvious clinical benefit, in 
which case the patient could be allowed to remain on 
treatment after having discussed and agreed on the case with 
the Sponsor(s)).


Patient or patient’s representative refusal and/or non 
compliance with study requirements.
Intercurrent serious illness. 
Protocol deviation with an effect on the risk/benefit ratio of 
the clinical trial.
Treatment delay > 2 weeks or impossibility to administer 
both Day 8 and Day 15 doses on a given cycle (except in 
case of clear clinical benefit, with the Sponsor(s)’s approval).
Requirement of > 2 dose reductions (except in case of clear 
clinical benefit, with the Sponsor(s)’s approval).
Administrative reasons or Sponsor(s)’s decision.
Any subsequent therapies for the patients may be provided off-
study according to the Investigator’s criteria.
REPLACEMENT OF 
PATIENTS
Patients must be replaced if they are considered not evaluable for 
efficacy, i.e., if they are withdrawn from the study due to 
significant 
clinical 
deterioration 
of 
unknown 
reason, 
hypersensitivity reactions, patient refusal or unrelated AEs 
without any tumor assessments after the start of study treatment.
PLANNED TRIAL 
PERIODS
The total duration of the study will be approximately 36 months, 
including about 24 months of active enrollment.
Consenting patients will be followed until death, or until the date 
of study termination, whichever occurs first. 
Planned start date (first patient on study): 1Q11.
Planned enrollment period: 24 months.
Planned study termination will occur:

Depending on the development/extension of the trial: 
o
Three months after the last treatment visit of the 
last evaluable patient if the study ends at the first 
stage.
o
Six months after the last treatment visit of the 
last evaluable patient, if the study proceeds to 
the second stage.

Or 12 months after the last patient included in the study, 
if neither of the above have occurred.


SCHEDULE OF ASSESSMENTS AND PROCEDURES
PRE-
TREATMENT
TREATMENT
(Cycle 1 and further cycles)
FOLLOW-
UP (1)
Study day
D1
D8
D15
D22
D28
(=D1 
of next 
cycle)
End of 
treatment
(last dose 
+ 30 days)
Every 3 
months
Study visit window
-
Within 2 days of pre-specified date
±1 week
Written informed 
consent
Before any study  
procedure
-
-
-
-
-
-
-
Medical history
D–28 to D1
-
-
-
-
-
-
-
Complete physical 
examination (2)
D–28 to D1

Repeat if clinically indicated


ECOG-PS(2)
D–28 to D1

Repeat if clinically indicated


Vital signs (2, 3)
D–28 to D1



-



Weight, height and BSA
D–28 to D1

Repeat if clinically 
indicated 

Repeat if clinically 
indicated 
Hematology (4)
D–7 to D1



-



Biochemistry A (4, 5)
D–7 to D1



-



Biochemistry B (6) 
D–7 to D1

Repeat if clinically 
indicated



Calculated creatinine 
clearance (6)
D–7 to D1

Repeat if clinically 
indicated

Repeat if clinically 
indicated
Pregnancy test (7)
D–14 to D1
Every 4 weeks during treatment and 3 months after last dose 
administration
ECG (5, 8)
D–28 to D1
Before each Zalypsis® infusion


LVEF (ECHO) (5, 9)
D–28 to D1
Every other cycle


Concomitant diseases 
and treatments
D–28 to D1
Throughout the “on-treatment” period

Adverse events
NA
Throughout the “on-treatment” period

Tumor assessment (10)
D–28 to D1
Every other cycle (± 1 week) until 
PD


Pharmacokinetics (11)
D1


-
-
-
-
-
Pharmacodynamic and 
pharmacogenomic 
samples, if patient 
consented (12, 13)
D–28 to D1

-
-
-

-
-
Other tests 
D–28 to D1
When indicated, according to the 
clinical and laboratory context


See notes on next page →


SCHEDULE OF ASSESSMENTS AND PROCEDURES (CONTINUED)
On-treatment period = from first infusion of Zalypsis® to End of treatment.
End of treatment = 30 days after the day of last dose of study drug administration, unless the patient starts a new 
antitumor therapy or dies (whichever occurs first), in which case the date of administration of this new therapy or the 
date of death will be considered the date of end of treatment.
1.
Patients withdrawn with Zalypsis®-related ongoing adverse events (AEs) must be followed up with the 
appropriate tests until event resolution. 
2.
Complete physical examination, ECOG PS, vital signs, and weight must be repeated on Day 1 of Cycle 1, prior 
to administration of the first Zalypsis® infusion.
3.
Before any Zalypsis® infusion.
4.
Repeat on Day 1 of Cycle 1 prior to treatment with Zalypsis®, if the treatment is administered more than one 
week after the pre-treatment tests. In subsequent administrations, repeat (with a −2 day window) before 
Zalypsis® infusion. If laboratory abnormalities grade  3 occur (according to the National Cancer Institute 
Common Terminology Criteria for Adverse Events [NCI-CTCAE] v. 4.0), the appropriate test(s) must be done 
at least every 2-3 days until recovery recovery to grade ≤ 1 or baseline. In case of febrile neutropenia (any grade) 
absolute neutrophil count (ANC) determination must be repeated every day until resolution.
5.
Follow-up of cardiac assessments (including ECG and LVEF) should be performed at three and six months after 
end of treatment. All troponin I determinations, ECGs and LVEFs will be reviewed by external cardiologists.
6.
Repeat on Day 1 of Cycle 1 whenever clinically indicated. If laboratory abnormalities grade  3 occur 
(according to the NCI-CTCAE v. 4.0), the appropriate test(s) must be done at least every 2-3 days until recovery 
to grade ≤ 1 or baseline.
7.
Pregnancy test, if applicable. Repeat on Day 1 of Cycle 1. 
8.
Electrocardiogram (ECG) must be repeated prior to the administration of each Zalypsis® infusion and if 
clinically indicated. 
9.
LVEF by ECHO to be performed every other cycle (with a 1-week window) and if clinically indicated. 
10. Computed tomography (CT)-scan or magnetic resonance imaging (MRI) of all evaluable sites of disease, as per 
Response Evaluation Criteria In Solid Tumors (RECIST) v. 1.1, within four weeks prior to the first dose of 
Zalypsis®. Documentation of progressive disease (PD) is mandatory at study entry.
11. First two infusions in Cycle 1. 
12. Circulating tumor cells (CTCs) will be collected on Day 1 before each Zalypsis® infusion for the first six cycles, 
and every three cycles thereafter. 
13. Paraffin-embedded tumor tissue (obtained at diagnosis of the tumor) can be collected any time after registration. 
Hematology: differential WBC (neutrophils, lymphocytes), hemoglobin and platelets.
Biochemistry A: liver function tests (ALT, AP, AST, LDH, total bilirubin), creatinine, CPK, troponin I, glucose, 
serum electrolytes (Na+, Cl-, K+, Ca++ and Mg++).
Biochemistry B: total protein and albumin.
AE, adverse event; ALT, alanine aminotransferase; ANC, absolute neutrophil count; AP, alkaline phosphatase; AST, 
aspartate aminotransferase; CPK, creatine phosphokinase; CPK-MB, creatine phosphokinase-fraction MB; CT, 
computed tomography; CTCs, circulating tumor cells; ECG, electrocardiogram; ECHO, echocardiogram; ECOG, 
Eastern Cooperative Oncology Group Performance Status; LDH, lactate dehydrogenase; LVEF, left ventricular 
ejection fraction; MRI, magnetic resonance imaging; NA, not applicable; NCI-CTCAE, National Cancer Institute 
Common Terminology Criteria for Adverse Events; PD, progressive disease; PK, pharmacokinetics; RECIST, 
Response Evaluation Criteria In Solid Tumors; WBC, white blood cells.


LIST OF ABBREVIATIONS AND DEFINITION OF TERMS
AE(s)
Adverse Event(s)
ALT 
Alanine Aminotransferase
ANC
Absolute Neutrophil Count
AP
Alkaline Phosphatase
ASCO
American Society of Clinical Oncology
ASCT
Autologous Stem Cell Transplantation
AST 
Aspartate Aminotransferase
βhCGs
Beta Human Chorionic Gonadotrophins
BSA
Body Surface Area
Cmax
Maximum Plasma Concentration
CNS
Central Nervous System
CPK 
Creatine Phosphokinase
CR
Complete Response
CRF
Case Report Form
CT
Computerized Tomography
CTC(s)
Circulating Tumor Cell(s)
D
Day
DSB(s)
Double Strand Break(s)
DNA
Deoxyribonucleic Acid
DOR
Duration of Response
ECG 
Electrocardiogram
ECHO
Echocardiogram
ECOG
Eastern Cooperative Oncology Group
EFT
Ewing’s Family of Tumors
EGFR
Epidermal Growth Factor Receptor
EI-CESS
European Intergroup Cooperative Ewing Sarcoma Studies
EOE
Extraosseous Ewing’s Sarcoma
EOI
End of Infusion
FISH
Fluorescence In Situ Hybridization
G-CSF
Granulocyte Colony Stimulating Factor
GCP
Good Clinical Practice
GM-CSF
Granulocyte-Macrophage Colony Stimulating Factor
GMT
Greenwich Meridian Time
h
Hour(s)
Hb
Hemoglobin
hERG
Human ERG Potassium Channel 
HBV
Hepatitis B virus
HCV
Hepatitis C virus
HIV
Human Immunodeficiency Virus
HRR
Homologous Recombination Repair
IB
Investigator’s Brochure
IC50
Half-maximal Inhibitory Concentration
ICH
International Conference on Harmonization
IEC
Independent Ethics Committee
IMP
Investigational Medicinal Product
IRB
Institutional Review Board
IUD
Intrauterine Contraceptive Device
i.v.
Intravenous(ly)
LDH
Lactate Dehydrogenase
LVEF
Left Ventricular Ejection Fraction
MedDRA
Medical Dictionary for Regulatory Activities
MoA
Mechanism of Action
MRI
Magnetic Resonance Imaging
mRNA
Messenger Ribonucleic Acid
MTD
Maximum Tolerated Dose


NA
Non Available/Non Applicable
NCI
National Cancer Institute
NCI-CTCAE
National Cancer Institute Common Terminology Criteria for Adverse Events
NER
Nucleotide Excision Repair 
NYHA
New York Heart Association 
OR
Objective Response
ORR
Overall Response Rate
OS
Overall Survival
RT-PCR
Retrotranscriptase Polymerase Chain Reaction
PD
Progressive Disease
PEN
Polyethylene Naphthalate
PFS
Progression-Free Survival
PGx
Pharmacogenomic(s)
PK
Pharmacokinetics
PM00104
Zalypsis
PNET
Primitive Neuroectodermal Tumor
PR
Partial Response
PS
Performance Status
PTEN
Phosphatase and Tensin Homolog
q3wk
Every Three Weeks
q4wk
Every Four Weeks
q.d.
Every Day
qRT-PCR
quantitative Real-Time Retrotranscriptase Polymerase Chain Reaction
RD
Recommended Dose
RECIST
Response Evaluation Criteria In Solid Tumors
RNA
Ribonucleic Acid
RT
Radiotherapy 
RT-PCR
Retrotranscriptase-Polymerase Chain Reaction
SAE(s)
Serious Adverse Event(s)
SD
Stable Disease
SOI
Start of Infusion
TTP
Time to Progression
ULN
Upper Limit of Normality
U.S./USA
United States/United States of America
WBC
White Blood Cells
WMA
World Medical Association


1.
INTRODUCTION
1.1.
EWING’S FAMILY OF TUMORS (EFT)
The Ewing’s family of tumors, which include Ewing’s sarcoma, primitive 
neuroectodermal tumor (PNET), Askin’s tumor of the chest wall and extraosseous 
Ewing’s sarcoma (EOE), are a group of tumors derived from the same primordial bone 
marrow-derived mesenchymal stem cell and have in almost all instances a clonal 
translocation in the long arms of chromosomes 11 and 22 (1).
The median age of patients with EFT is 15 years, and more than 50% are adolescents. 
EFTs are rare in adults older than 30 years. Based on data from 1426 patients entered 
into the European Intergroup Cooperative Ewing Sarcoma Studies (EI-CESS), 59% of 
patients were male and 41% female. Primary sites included lower limbs (41%), pelvis 
(26%), chest wall (16%), upper limbs (9%), spine (6%), and skull (2%). For EOE, the 
most common sites are trunk (32%), limbs (26%), head and neck (18%), 
retroperitoneum (16%) and other sites (9%) (2).
The prognosis of EFT varies depending on primary tumor site, presence of metastases 
and tumor size. The overall 5-year disease-free survival rate for localized Ewing’s 
sarcoma treated with surgery, radiation and multi-agent chemotherapy is 65-76% (3). 
However, the 5-year disease-free survival rate drops to 30% if metastases are present at 
diagnosis. Limited improvement of survival rates has been achieved for metastatic 
Ewing’s sarcoma. Similarly, relapsed patients have a poor outcome, with very common 
failure to second-line therapy and low 5-year survival rates (13%) (4).
1.1.1.
Therapeutic approach for EFT
A multidisciplinary approach, including surgery, chemotherapy, and radiotherapy (RT) 
is mandatory. Multi-agent chemotherapy is essential for Ewing's sarcoma due to the 
high risk of micrometastatic disease. Multi-drug chemotherapy for EFT always includes 
vincristine, 
doxorubicin, 
ifosfamide, 
and 
etoposide. 
Most 
protocols 
use 
cyclophosphamide as well and certain protocols incorporate dactinomycin. Duration of 
primary chemotherapy ranges from six months to one year (5).
In high-risk cases of Ewing’s sarcoma, myeloablative chemotherapy followed by 
autologous stem cell transplant (ASCT) has been used as consolidation therapy. 
Although better survival than expected was reported with this approach, large 
prospective trials are needed to properly evaluate the potential utility of this therapy in 
Ewing’s sarcoma patients (6). 
The management of relapsed or recurrent disease is not standardized and, in most cases, 
consists of different combinations of the same agents used as adjuvant therapy. Patients 
are often treated with more than one regimen to reduce disease burden to the minimum. 
Ifosfamide and etoposide are active in treating Ewing’s tumor of bone and should be 
considered for patients who have not previously received these agents. Combinations 
such as cyclophosphamide plus topotecan and irinotecan plus temozolomide have also 
been active in patients with recurrent or refractory disease. Radiotherapy of lung and 
bone, and surgical removal of metastases are also included in the management of these 
patients. However, patients with relapsed disease have a poor outcome; failure to 
second-line therapy is very common, with 5-year survival rates around 13% (4). In 
addition, the toxicity, morbi-mortality, and long-term complications of these agents 


(cardiac, renal, pulmonary, gonadal and secondary neoplasias) are considerable. 
Therefore, there is an urgent need for new therapeutic agents with different mechanisms 
of action to manage this patient population.
1.2.
ZALYPSIS® (PM00104)
PM00104 (Zalypsis®) is a new synthetic alkaloid that was selected for clinical 
development as an antineoplastic agent because of its broad in vitro cytotoxic activity 
against human solid and non-solid tumor cell lines, its in vivo activity in human tumor 
xenografts, as well as an acceptable non-clinical toxicology profile. The main bases for 
the preclinical and clinical development of PM00104 are shown here, although 
complete details and references can be found in the Investigator’s Brochure (IB), 
provided in a separate folder.
1.2.1.
Chemical Structure and Formulation
PM00104 (Figure 1) is currently produced by chemical synthesis.
Figure 1. Chemical structure of PM00104.
N
N
Me
Me
OMe
HO
Me
OH
O
O
AcO
NH
O
CF3
H H
H
1
5
3
6
7
8
11
12
14
16
2
3
3
1
6
Molecular formula: C37H38F3N3O8.; Molecular weight: 709.708.
1.2.2.
Mechanism of Action
Preliminary data on mechanism of action (MoA) suggest that PM00104 exerts effects 
on the cell cycle, and displays deoxyribonucleic acid (DNA)-binding properties as well 
as transcriptional inhibition (7).
Structure-activity relationship studies have led to the notion that the binding of 
PM00104 to DNA is a critical event in its cytotoxic action, but that an additional non-
DNA target may be required to elicit an optimal antitumor response.
PM00104 displays strong inhibition of the transcriptional response, with a similar 
pattern at all the concentrations studied. In addition, PM00104 strongly inhibits the 
activation of the transcription of other genes such as MDR1, a gene critically involved 
in resistance to many chemotherapeutic agents, without affecting constitutive 
transcription. More details on MoA can be found in Section 1.2.6.1.


1.2.3.
Preclinical Data
Zalypsis® has demonstrated antiproliferative in vitro activity against a broad spectrum 
of human solid and non-solid tumor types (Table 1). Regarding solid tumors, Zalypsis®
exhibited in vitro antitumor activity (i.e., IC50  10-8 M) against representative cell lines 
of bladder, colon, gastric, kidney, melanoma, pancreas, prostate, sarcoma, and thyroid; 
similar activities were noted against leukemia and lymphoma as representatives of non-
solid tumors.
Zalypsis® also demonstrated in vitro antitumor activity, although to a lesser extent (i.e., 
IC50 ranging from 10-7 to 10-6 M), in representative cell lines of breast, lung, sarcoma 
(SK-LMS-1 and SW-684), and lymphoma (U937). In vitro activity (i.e., 10-6 to 10-5 M) 
was not seen, however, in a representative ovarian cell line (SK-OV-3).
Table 1. In vitro activity of Zalypsis®
Type 
Tumor
Cell line
IC50 (M)
Bladder
5637
8.6·10-09 – 1.1·10-09
BT-474
6.2·10-07 – 8.2·10-07
Breast
MX-1
4.7·10-06 – 8.5·10-06
Colon
HT-29
1.1·10-08 – 7.7·10-07
Gastric
Hs746T
2.1·10-08 – 5.5·10-11
Kidney
768-O
1.0·10-08 – 2.1·10-09
Liver
SK-HEP-1
2.2·10-07 – 2.3·10-07
Lung
A459
3.0·10-06 – 4.8·10-06
Melanoma
SK-MEL-28
1.7·10-08 – 2.0·10-09
Ovarian
SK-OV-3
1.7·10-06 – 1.5·10-05
Pancreas
PANC-1
7.9·10-08 – 2.3·10-09
DU-145
2.7·10-06 – 4.2·10-06
LNCaP
7.5·10-08 – 1.1·10-08
Prostate
PC-3
4.2·10-08 – 6.8·10-07
A-673
1.6·10-12 – 2.0·10-12
CHSA
1.8·10-08 – 1.0·10-09
OSA-FH
1.5·10-08 – 4.1·10-07
SK-LMS-1
8.7·10-07 – 2.2·10-06
Sarcoma
SW-684
2.7·10-06 – 3.7·10-06
Solid
Thyroid
SW-579
1.1·10-08 – 2.3·10-09
HL-60
5.4·10-08 – 8.6·10-08
Leukemia
K-562
 9.4·10-09 – 1.3·10-08
H9
3.7·10-11 – 2.0·10-9
MC116
3.9·10-08 – 2.5·10-08
Non-solid
Lymphoma
U937
1.4·10-07 – 2.1·10-07
Source: Investigator’s Brochure.


In vivo tumor profiling of Zalypsis® was obtained by using a panel of six human tumor 
types, i.e., breast, colon, gastric, ovarian, prostate, and renal. The resulting tumor 
susceptibility was analyzed in xenografts grown in athymic mice when Zalypsis® was 
administered as single bolus intravenous (i.v.) injections (data from the IB).
Table 2. Antitumor activity of Zalypsis® in human athymic mouse xenografts
Tumor type,
cell line
Route and Schedule
Dose levels 
mg/kg (mg/m2)
Antitumor Effect 
(Optimal Day)
Breast,
MX-1
i.v., qdx1
0.75 (2.25)
-64% ∆T/∆C (Day 9)*
Colon,
HT-29
i.v., qdx1
0.75 (2.25)
47% ∆T/∆C (Day 7)§
Kidney,
MRI-H-121
i.v., qdx1
0.75 (2.25)
7% ∆T/∆C (Day 6)*
Ovary,
SK-OV-3
i.v., qdx1
0.75 (2.25)
60% ∆T/∆C (Day 6)
Prostate,
PC-3
i.v., qdx1
0.75 (2.25)
2% ∆T/∆C (Day 7)*
Stomach,
MRI-H-254
i.v., qdx1
0.75 (2.25)
14% ∆T/∆C (Day 15)*
* p < 0.05, statistically significant compared to control cohort
§ p < 0.06, trend to significance
T/C = % ratio of treated versus control tumor volumes; i.v., intravenous, qd, every day.
Zalypsis® demonstrated statistically significant antitumor activity against breast, gastric, 
and renal malignancies (Table 2). In colon tumors, only low significance was observed 
after Zalypsis® administration as a single i.v. bolus for the sample size tested (i.e., n = 
5). With regard to gender-specific malignancies, Zalypsis® significantly inhibited 
prostate tumors, but was inactive against ovarian tumors. In summary, Zalypsis®
demonstrated strong antitumor activity in breast, gastric, prostate, and renal, but had a 
more moderate antitumorigenic profile against colon.
The antitumor effectiveness of Zalypsis® was further analyzed in xenografts using the 
susceptible human breast tumor MX-1 after determination of single-day (i.e., qdx1) 
maximum tolerated dose (MTD) levels in mice, i.e., 0.75 mg/kg. Specifically, 
decreasing total doses were administered on a qdx1 i.v. bolus schedule (i.e., 1.0, 0.75, 
and 0.5 MTDqdx1, respectively, each on Day 0), while increasing total levels were tested 
using a five-consecutive-day i.v. administration regimen (i.e., 1.0, 1.25, and 1.67 
MTDqdx1, respectively, each on Days 0-4). Interestingly, the qdx5 i.v. schedule appeared 
to have an optimal antitumor effect for Zalypsis® (data from the IB). 
Zalypsis®, given by i.v. injection, produced toxicological effects typical of cytotoxic 
antitumor agents. Tissues containing cells with a high turnover rate were especially 
targeted in rats and dogs, i.e., bone marrow, reticuloendothelial system, and 
gastrointestinal tract, as well as the liver, the reproductive system and lesions in the 
injection site. Most toxicities were reversible or in repair at the end of an acute toxicity 
evaluation. Toxicological effects were more severe when the compound was given as a 
multicycle 24-hour infusion in rats and dogs. In the fractionated dose studies performed 
so far, Zalypsis® was well tolerated when administered to rats and dogs at levels based 
on dividing the MTD, calculated after single administration by a factor of five or three, 
for five consecutive days or three consecutive weeks, respectively. Zalypsis® displayed 


less liver toxicity when administered using fractionated daily doses compared to single 
bolus administration. In rats receiving Zalypsis® at MTD levels (single administration), 
increases in liver function markers as well as histology findings related to liver injury 
were seen. However, these findings were not detected in those animals which received 
an equivalent total dose but in a fractionated five consecutive daily schedule of 
Zalypsis®. Regardless of the dosing schedule, toxicity related to hematopoietic and 
thymus systems were observed.
With respect to the safety pharmacology of Zalypsis®, none of the observations 
regarding the alterations triggered by Zalypsis® on either neurotoxicity, cardiovascular 
or respiratory function, raised any concern. For multicycle studies, Zalypsis® was well 
tolerated in rats and dogs at the MTD values when administered as a bolus. However, 
the administration of two (on Days 1 and 15) 24-hour infusions of Zalypsis® in rats 
resulted in deaths occurring at the higher dose levels studied. Histology evaluation 
revealed hemorrhage, necrosis and mineralization of the heart in many of the early dead 
animals, accompanied by degeneration in the animals treated at the highest doses. 
Zalypsis® appears to be more toxic after multiple 24-hour administration compared to 
either single 24-hour or single/multiple bolus administration in rats and dogs. 
In the human ERG potassium channel (hERG) assay, after a 5-min exposure of HEK293 
cells stably transfected with hERG complementary DNA (cDNA) to a concentration of 
15 µM of Zalypsis®, inhibition of the hERG tail current was complete. Increasing 
concentrations of Zalypsis® (0.01, 0.03, 0.1, 0.3 and 3 M) produced changes which 
were fitted into a sigmoidal function, with a calculated IC50 of 0.4 M (0.28 g/ml), 
well above the Cmax found in animals. 
As a measure of precaution and to ensure the safety of the patients, closer cardiac 
monitoring was performed during the phase I clinical development.
1.2.4.
Metabolism
In the early phases of drug development, several in vitro studies were carried out to 
assess the biotransformation pathways of Zalypsis® in humans and other animals. These 
studies indicated that Zalypsis® underwent extensive hepatic microsomal-mediated 
metabolism in all animal species, including man. Measurement of Zalypsis® in urine 
samples was not possible due to the instability of this drug in this medium (8).
1.2.5.
Clinical Data
The clinical development program includes four phase I clinical trials aimed to assess 
five different schedules of administration in patients with solid malignancies or 
lymphoma for which no standard therapy would reasonably be expected to result in cure 
or palliation. 
One hundred and forty four patients have been included in phase I trials with Zalypsis®
as of December 2009. Prophylactic antiemetic therapy with dexamethasone plus 
ondansetron or similar agents before the infusions was mandatory after the emetic 
events and dose-limiting toxicities (DLTs) were observed in the clinical trial PM104-A-
001-04 (Table 3). A central catheter is mandatory since cases of infusion site reactions 
were also reported.
With respect to hematological events, decrease in blood cell counts have been observed 
(expected according to preclinical toxicology results), especially at the highest dose 
levels. In most cases, these hematological events were mild to moderate, transient and 


not associated with clinical manifestations, such as fever, infection or hemorrhages.
With respect to biochemical events, severe increases in liver transaminases were rare 
and no grade 4 cases were reported. The laboratory abnormalities usually recovered 
within few days and no concomitant symptoms were reported. 
As previously mentioned, closer cardiac monitoring was included in the clinical phase I 
program as a measure of precaution due to some findings in preclinical studies. Cardiac-
related disorders found in these phase I studies have consisted of elevations of cardiac 
troponin 
I 
without 
other 
symptoms 
or 
relevant electrocardiogram 
(ECG)/echocardiogram (ECHO) alterations. Four patients treated in the study PM104-
A-004-05, which evaluated the same schedule than that to be used in the current phase 
II trial (i.e., 1-hour Day 1, 8 and 15 every four weeks [q4wk]) had grade 4 troponin I 
increase at the highest dose levels: 2.0 mg/m2 (n=2), 2.5 mg/m2 (n=1) and 3.0 mg/m2
(n=1). None of them had cardiac AEs, abnormal ECG or decrease in left ventricular 
ejection fraction (LVEF) at the same time than troponin I increase, except one patient 
treated with 3.0 mg/m2 who had grade 1 sinus tachycardia and a slight shortening of the 
QT interval. These events were considered “not relevant” by the Investigators and by an 
external cardiologist who evaluated these cases. ECGs performed as per study protocols 
before and after each infusion of Zalypsis® have been reviewed by an external 
cardiologist and no evidence of cardiotoxicity has been reported. However, close 
clinical and investigational monitoring of cardiac safety will be performed in the current 
study with Zalypsis®.
No toxic deaths have occurred in the phase I studies with Zalypsis®.
The recommended dose (RD) for phase II studies, 2 mg/m2, to be used in the current 
clinical trial (PM104-B-003-10) was determined by the study PM104-A-004-05 which 
used the same schedule (d1, d8 and d15; q4wk). A summary of the results of this 
finalized study can be found in Table 3.


Table 3. Summary of the phase I study PM104-A-004-05
Dose escalation 
cohorts
Dose 
level
Dose 
(mg/m2)
Patients included /patients 
with DLTs 
(n=49)
DLTs observed per patient
I
0.07
1 / 0
.
II
0.15
1 / 0
.
III
0.3
1 / 0
.
IV
0.6
 4 / 0*
.
V
0.9
3 / 0
.
VI
1.35
3 / 0
.
VII
2.0
(RD)
24 / 1

Grade 4 lipase increase and grade 3 
diarrhea NOS
VIII
3.0
(HDR)
4 / 2

Grade 4 fatigue

Grade 3 fatigue, grade 4 neutropenia 
lasting 
> 
5 
days, 
grade 
4 
thrombocytopenia and grade 3 cardiac 
troponin I increase**
IX 
2.5
(MTD)
8 / 4

Grade 1 thrombocytopenia and grade 2 
neutropenia with two dose omissions

Grade 4 febrile neutropenia

Grade 4 cardiac troponin I increase**

Grade 3 fatigue
The shadowed row corresponds to data from the RD.
*At dose level IV, due to grade 3 transaminases (ALT and AST) increase in patient #104 (early 
disease progression) and drug-related grade 2 neutropenia in patient #107, in the subsequent dose 
levels cohorts were expanded to at least three patients and dose escalation was reduced to 50%.
**Two episodes of grade 3/4 cardiac troponin I increase occurred (patients #220 and #128), both were 
considered by the investigators as DLT despite not meeting all criteria established for DLT definition 
in the protocol (i.e., they were not associated with any evidence of cardiac damage by ECG or ECHO).
ALT, alanine aminotransferase; AST, aspartate aminotransferase; DLT, dose-limiting toxicity; ECG, 
electrocardiogram; ECHO, echocardiography; HDR, highest dose reached; MTD, maximum tolerated 
dose; NOS, not otherwise specified; RD, recommended dose.
1.2.5.1. Clinical Trial PM104-A-004-05
Forty nine patients (29 males and 20 females) with malignant solid tumors have been 
enrolled and treated at the participating institutions (Institut Gustave Roussy, Paris, 
France, and Newcastle General Hospital-Northern Centre for Cancer, Newcastle, United 
Kingdom). The median age of the patients was 58 years (range, 22-76 years). 
Table 3 summarizes the dose escalation, the patients treated at each cohort and the 
DLTs observed. The patients were distributed in nine dose levels. The starting dose was 
0.07 mg/m2. According to an accelerated design with escalating doses of Zalypsis®, one 
patient per dose was treated and dose was escalated by 100% from dose level I (0.07 
mg/m2) to dose level III (0.3 mg/m2). The first patient treated at dose level IV (0.6 
mg/m2) had non-drug related grade 3 ALT and AST increase due to early disease 
progression. For safety reasons, this dose level was expanded to four patients. Another 
patient at this dose level had grade 2 drug-related neutropenia in the first treatment 
cycle. Due to the aforementioned events (nevertheless, not evaluated as DLTs), the 


study continued with expanded cohorts of at least three patients and with dose 
escalation by 50% in dose level V (0.9 mg/m2) and afterwards.
No DLTs were observed from dose level I (0.07 mg/m2) to dose level VI (1.35 mg/m2). 
After four consecutive dose increases by 50%, the first two DLTs were reported in dose 
level VIII (3.0 mg/m2, the highest dose reached). Upon agreement with the 
investigators, an intermediate dose between this and the next lowest level (dose level 
VII; 2.0 mg/m2) was proposed, and subsequent patients were enrolled and treated at a 
dose of 2.5 mg/m2 (dose level IX). Four of eight patients treated with this dose had 
DLTs; therefore, 2.5 mg/m2 was declared to be the MTD and 2.0 mg/m2 (one DLT in 24 
treated patients) was defined as the RD.
The tumor types included in this trial were gastrointestinal: colorectal adenocarcinoma 
(n=14), gastric carcinoma (n=5), esophagus carcinoma (n=4), pancreas adenocarcinoma 
(n=3), cholangiocarcinoma (n=2) and anal carcinoma (n=1); genitourinary: ovarian 
adenocarcinoma (n=2), prostate adenocarcinoma (n=2) and bladder carcinoma, cervix 
carcinoma and endometrial adenocarcinoma (n=1 each); respiratory: pleural 
mesothelioma (n=3) and non-small cell lung cancer (NSCL) (n=2); and others: 
malignant melanoma (n=4), soft tissue sarcoma (n=3) and lachrymal adenoid carcinoma 
(n=1).
At the recommended dose (RD) (2.0 mg/m2), the total number of cycles administered 
was 24, with a median number of cycles administered per patient of 2 (range, 1-5 
cycles). The median cumulative dose was 2.9 mg/m2 (range, 0.7-8.0 mg/m2), the median 
dose intensity was 1.2 mg/m2 (range, 0.15-2.6 mg/m2) and the median relative dose 
intensity was 77.9% (range, 33.2-103.2%).
1.2.5.1.1.
Safety Data
The most common treatment-related AEs that occurred at the RD were nausea (87.5% 
of patients/77.4% of cycles), vomiting (83.3% of patients/62.3% of cycles) and fatigue 
(75.0% of patients/62.3% of cycles).
Five patients treated at the RD had six drug-related serious adverse events: grade 3 
nausea and grade 3 neutropenic infection (both occurring in one patient); grade 3 
diarrhea, grade 3 tumor flare, grade 3 tumor pain, and grade 1 pyrexia (one patient 
each). 
No cardiac events or troponin increases were observed at the recommended dose.  
The most frequent hematological abnormality at the RD was anemia, which was 
observed in all patients, although in most cases of mild degree. Nine patients (37.5%) 
worsened their hemoglobin values to grade 2 and only one patient (4.2%) reached grade 
3 during one cycle. Other severe hematological toxicities at the RD consisted of grade 3 
thrombocytopenia in one patient (4.2%) during one cycle, grade 3 leukopenia (8.3%) 
and grade 3/4 neutropenia (8.3%). These hematological abnormalities were reversible 
(lasted 1-7 days) and non-cumulative.
The most common severe non-hematological laboratory disorder at the RD was grade 3 
transaminase increase (ALT, 4.2%; AST, 8.3%), followed by grade 3 AP increase 
(8.3%) and grade 3 total bilirubin increase (4.2%). No cases of grade 4 biochemical 
abnormalities were found at the RD. All cases of severe non-hematological toxicities 
found at this dose level were reversible and asymptomatic.


1.2.5.1.2.
Efficacy Data
Four disease stabilizations lasting longer than 3 months were observed at different dose 
levels in patients with cervix carcinoma, colorectal adenocarcinoma, lachrymal adenoid 
carcinoma and bladder carcinoma (Table 4). 
Table 4. PM104-A-004-05. Stable disease lasting ≥ 3 months.
Tumor type
Gender
Age
(years)
Zalypsis®
dose 
level
(mg/m2)
No. of 
prior 
regimens
Agent/s of last 
prior therapy
Best 
response 
to prior 
treatment 
/ TTP 
(months)
No. of 
Zalypsis®
cycles
received 
TTP 
with 
Zalypsis®
(months)
Cervix 
carcinoma
Female
51
II
(0.15)
1
Cisplatin
NA/NA
4
3.4
Colorectal 
adenocarcinoma
Male
57
V
(0.9)
6
Erbitux/irinotecan
SD/5.9
5
5.1
Lachrymal 
adenoid 
carcinoma
Male
70
VI
(1.35)
2
Imanitib
SD/1.8
8
9.5
Bladder 
carcinoma
Male
45
IX 
(MTD)
(2.5)
2
5-fluorouracil/ 
cisplatin/epirubicin
 SD/14.0
5
6.5
NA, not available; MTD, maximum tolerated dose; TTP, time to progression.
In addition, a phase II clinical trial (PM104-B-001-09) has been started to evaluate the 
antitumor activity of Zalypsis® administered with the same dose and schedule (1-h d 1, 
8 and 15 q4wk) to that which will be used in the current study, in patients with 
advanced and/or metastatic endometrial or cervical cancer previously treated with one 
line of systemic chemotherapy. As of 31 May 2010, 16 patients have been recruited in 
this study.
1.2.5.2. Pharmacokinetic Data
The main pharmacokinetic (PK) characteristics of Zalypsis® are: prolonged half life (up 
to 50 hours), wide distribution (around 800 l), high interpatient variability and linear 
PK, except for doses higher than 3000 µg/m² infused in 1 hour.
1.2.6.
Pharmacogenomic Data
For the past 30 years medical oncologists have focused on optimizing the outcome of 
cancer patients, developing new antitumor agents and defining new prognostic factors 
as well as integrating more effective supportive care measures. However, clinical 
anticancer strategies indicate that conceptually active therapies benefit just a small 
proportion of patients. As a consequence, a large cohort of patients needs to be exposed 
to antitumor treatments to obtain benefit in just a fraction of them.
Molecular targeted therapies and personalized cancer therapies are concepts that are 
raising expectations in anticancer drug development as a consequence of the genomics 


technology developed and our improved understanding of cancer at the molecular level. 
Pharmacogenomic (PGx) studies are aimed at identifying prognostic biomarkers that 
can help to define subpopulations of patients who will benefit from a particular therapy. 
These molecular markers of response to the drugs are not exclusive of the so-called 
“targeted therapies”, but have also been identified in widely used cytotoxic agents. 
Representative examples include the relation between thymilydate synthase mRNA 
expression and response and survival with antifolates (9), beta tubulin III mRNA levels 
and response to tubulin interacting agents (10), Phosphatase and Tensin Homolog 
(PTEN) methylation and resistance to irinotecan (11), and STAT3 overexpression and 
resistance to epidermal growth factor receptor (EGFR) interacting agents (12).
For the last five years, the Sponsor(s) have implemented a translational research 
program oriented to the identification of molecular markers correlated with the response 
to the anticancer agents in development. Based on the experimental studies that indicate 
that there is an increased sensitivity to trabectedin in tumors with efficient nucleotide 
excision repair (NER) and deficient homologous recombination repair (HRR) DNA 
repair machinery, the expression of a set of DNA repair genes was analyzed in a 
retrospective cohort of sarcoma patient tumor samples (13). The main result obtained 
was the identification of the levels of expression of the gene BRCA1 as a marker that 
correlates with longer progression-free survival (PFS) and overall survival (OS) after 
the treatment with trabectedin (14). This fact, which nowadays has been validated in 
samples from a larger cohort of patients, opens the possibility of identifying those 
patients that would benefit from the treatment of trabectedin in a prospective fashion.
These very promising results are the bases for a more exhaustive research for molecular 
markers of tumor response to treatment that will in the future allow a real personalized 
antitumoral medicine.
1.2.6.1. Rationale for the Pharmacogenomic Substudy Associated to PM104-B-
003-10
Zalypsis® is a novel synthetic antineoplastic agent currently in Phase II clinical 
development. It has strong antitumor activity in a wide variety of tumor lines in vitro
and in vivo. Preliminary in vitro data suggest that Zalypsis® has DNA binding 
properties, induces cell cycle arrest and inhibits transcription. Although the precise 
mechanism of action of Zalypsis® has not been fully elucidated, there are increasing 
experimental data of Zalypsis® antitumor activity. Three main molecular characteristics 
describe the antitumor activity of Zalypsis®: 
1. Zalypsis® is a DNA binder: Zalypsis® binds to the minor groove of DNA. This 
binding occurs in preferred trinucleotide sequences GC rich, preferably to GCG 
trinucleotide (from C. Bailey, unpublished PharmaMar internal report). The binding 
of Zalypsis® to the DNA produces a stabilization of the DNA duplex, with notable 
increase (13–18ºC) in the melting temperatures of DNA oligonucleotides containing 
either single or tandemly arranged binding sites (15). This stabilization could 
account for the need of the same DNA repair machinery that usually deals with 
inter-strand cross-links and involves proteins from both HRR and NER machineries 
(16).
2. Zalypsis® produces DNA double strand breaks (15): In cell lines, the Zalypsis®-
DNA adducts provoked DNA double-strand breaks (DSBs), evidenced by an 
increase in phospho--H2AX and phospho-CHK2. In addition, treatment of cells 


with Zalypsis® led to cell cycle delay in S phase, activation of the DNA damage 
checkpoint and apoptotic cell death. More interestingly, Zalypsis® has shown a 
potent anti-myeloma action on cell lines and patient plasma cells, as well as on 
xenografted myeloma cells in mice (7). The action of Zalypsis® in plasma cells 
involved cell cycle blockade and apoptosis. The latter occurred by both caspase-
dependent and independent routes. Zalypsis® provoked DNA DSBs, which were 
evidenced by an increase in phospho histone H2AX, causing up-regulation of p53 in 
myeloma cell lines bearing wild type forms of this protein. This increase in p53 was 
followed by augmented levels in p53-regulated proteins such p21, Noxa or Bak. 
Also, Schizosaccharomyces pombe containing a RAD51 mutation were found to be 
extremely sensitive to Zalypsis®, suggesting that the compound induces DSBs. 
3. Zalypsis® interferes with DNA repair: Experimental data reveal that the DNA 
damage repair machinery is essential to overcome Zalypsis® induced DNA damage 
and suggest that this damage is mainly due to DSBs. It has been described that the 
antiproliferative activity of Zalypsis® does not depend on transcription-coupled 
NER. Zalypsis® induces phosphorilated histone γ-H2AX foci with the same 
efficiency in NER-deficient or NER-proficient cells. Moreover, the formation of γ-
H2AX foci is replication dependent for Zalypsis®, effects suggestive of stalled 
transcription and replication forks (17). In addition, the dependency of the cytotoxic 
effects of the drug on an intact NER system have been studied on cells deficient in 
RAD13, which is the Schizosaccharomyces pombe counterpart of human XPG, a 
NER protein that has been shown to mediate, at least in part, the MoA of other 
agents in this class in human cells (18). RAD13 haploid deletion mutants were 
found to be as sensitive to Zalypsis® as wild-type cells indicating the independence 
of the cytotoxic effect of this compound to the lack of a functional nucleotide 
excision repair system. This evidence is in contrast with the pattern noted with other 
antitumors with DNA binding activity as trabectedin and cisplatin that are highly 
dependent on the activity of this repair pathway. The same study determined the 
sensitivity to Zalypsis® in a collection of 5000 haploid deletion mutants of the yeast 
Saccharomyces cerevisiae as a model. We have identified approximately 40 
hypersensitive mutants and about 90 mutants resistant to Zalypsis® activity. Among 
the genes which deletion produced sensitivity to Zalypsis® we found a set of genes 
involved in sensing/repairing DSB, such as components of the MRX complex, 
several members of the homologous recombination proteins of the Rad52 epistasis 
group (recruited to the sites of DNA damage specifically during S-phase and G2), 
the Swi/Snf complex and a chromatin remodeling complex conserved in humans. 
Swi/Snf inactivation results in inefficient DSB repair and increased DNA damage 
sensitivity as well as a large defect in γ-H2AX phosphorylation strain with a
deletion in SUMO ligase, Siz1 was one of the most resistant mutants to Zalypsis®. A 
role for this protein in the inhibition of homologous recombination at replication 
forks has been described recently, indicating again that the main lesions induced by 
Zalypsis® are DSBs.
4. Zalypsis® interferes with transcription: PM0104 inhibits the in vitro transcription of 
RNA both at the initiation and at the elongation phases (J.M. Egly personal 
communication). The inhibition of transcription induced by Zalypsis® is 10-fold 
more potent that induced by trabectedin. Studies conducted in a panel of sarcoma 
cell lines have identified a very significant correlation associating the expression of 
PDGFR-α and phosphorylation of c-kit with the in vitro sensitivity to Zalypsis®


(19). This association has been confirmed in a larger panel of cell lines of epithelial 
origin outside from the sarcoma indication, both in vitro and in vivo mice 
xenografts. Although it is not well understood how these data fit into the MoA of 
Zalypsis®, this strong correlation deserves a clinical confirmatory study.
Based on the available experimental data, it seems reasonable to develop studies to 
analyze the correlations between the tumor/patient and genes/proteins determinants in 
the efficiency/deficiency of the biological pathways shown above and the outcome of 
patients exposed to Zalypsis®. The ultimate goal has been the identification of 
molecular biomarkers present on patients that shall be prone to respond to Zalypsis® in 
order to implement a customized therapy in the future. 
1.3.
RATIONALE FOR THIS PHASE II EXPLORATORY TRIAL OF ZALYPSIS® IN EFT
Ewing’s sarcoma is an aggressive neoplasm of the bone and soft tissues that arises 
primarily in adolescence and young adulthood. It is characterized by the occurrence of 
non-random gene rearrangements between the EWS gene on chromosome 22q12 with 
various members of the ETS gene family (20, 21). Although the introduction of 
chemotherapy has significantly improved the chance of survival of non-metastatic 
patients, shifting the 5-year survival rate to around 50–60% (22-24), in the last two 
decades the survival rate of Ewing’s sarcoma patients has reached a plateau phase (25, 
26). Particularly, limited improvement of survival rates has been achieved for metastatic 
and relapsed Ewing’s sarcoma, with 5-year survival rates dropping to 13% in the latter; 
therefore, relapsed Ewing´s sarcoma represents an unmet medical need.
 There is evidence that sarcomas associated to translocations which deregulate 
transcription factors have high response rates to treatment with DNA binders such as 
trabectedin or Zalypsis® compounds.
o
Trabectedin has shown higher efficacy in the treatment of sarcomas having 
specific translocations producing deregulated transcription factors including 
FUS-CHOP myxoid/round cell liposarcoma (27-29) and Ewing’s sarcoma (27, 
30-32) although the specific mechanisms of action are still under investigation.
o
Interestingly, they share TET-family genes (EWS or FUS) as gene fusion 
components. The fusion proteins contain a DNA binding domain and a 
transcriptional activation domain under the control of a constitutively expressed 
promoter that makes the fusion protein a deregulated transcription factor.
o
Trabectedin has been described to impede the binding of FUS-CHOP fusion 
protein to some promoters regulated by the chimerical protein, interfering with 
the expression of downstream proteins (33).
o
Both Zalypsis® and trabectedin have been claimed to interfere with the 
transcriptional activity of EWS-FLI1, downregulating the expression of some 
EWS-FLI1 regulated genes such as NR0B1 (DAX1) (34).
o
Zalypsis® is a DNA binder that binds to DNA at the minor groove with a 
stronger effect in transcription regulation than in DNA repair as compared to 
trabectedin (7).
o
Zalypsis® shows an increased sensitivity compared to that of trabectedin in a 
panel of Ewing's sarcoma cell lines characterized by chromosomal translocations 
producing deregulated transcription factors (35). The effects of Zalypsis® and 
trabectedin on growth inhibition are shown in Figure 2.


o
Zalypsis® increased sensitivity in the Ewing’s sarcoma cell line panel is 
independent of the translocation/chimerical transcription factor variants. Wild-
type p53 cell lines are more sensitive to trabectedin than to Zalypsis® (35) (see 
Figure 3).
Figure 2. Effects of Zalypsis® and trabectedin on growth inhibition.
0
100
200
300
400
500
600
700
800
IC50 pM
EWS-ERGEWS-ERGEWS-ERG EWS-FLI1
1
EWS-FLI1
1
EWS-FLI1
1
EWS-FLI1
1
EWS-FLI1
1
EWS-FLI1
2
EWS-FLI1
2
EWS-FLI1
3
EWS-FLI1
3
rhabdo
TTC466
CADO-ES
RM82
SKNMC
A673
TC71
STAET1
WE68
SK-ES-1
SJRH
STAET2.1
A4573
RD-ES
Cell Line
IC50 GI Trabectedin vs PM0104
trabectedin pM
PM0104 pM
Increased sensitivity in terms of growth inhibition IC50 to Zalypsis® compared to trabectedin in a panel of 
Ewing’s sarcoma cell lines containing specific chromosomal translocations.
Figure 3. Effects of Zalypsis® and trabectedin on p53 impact
0
100
200
300
400
500
600
700
800
IC50 pM
wt
wt
wt
del EX2-4
2 bp ins 119
arg213Stop
cys176phe
cys277tyr
arg273his
arg273cys
EWS-ERG
EWS-FLI1 1 EWS-FLI1 1 EWS-FLI1 1 EWS-FLI1 1 EWS-FLI1 1 EWS-FLI1 2 EWS-FLI1 3
EWS-ERG
rhabdo
CADO-ES
WE68
STAET1
SKNMC
A673
TC71
SK-ES-1
STAET2.1
RM82
RD-ES
Cell line/p53 status
IC50 GI Trabectedin vs PM0104 p53 Impact
trabectedin pM
PM0104 pM
Increased sensitivity in terms of p53 IC50 to Zalypsis® compared to trabectedin in a panel of Ewing’s 
sarcoma cell lines containing specific chromosomal translocations.


2.
STUDY OBJECTIVES
2.1.
PRIMARY OBJECTIVE
•
To determine the antitumor activity of Zalypsis® administered as a 1-h i.v. infusion 
on d1, d8 and d15, q4wk, to patients with advanced and/or metastatic EFT.
2.2.
SECONDARY OBJECTIVES

To determine time-to-event efficacy parameters.

To characterize the safety profile and tolerability of Zalypsis® in patients with 
unresectable advanced and/or metastatic EFT.

To characterize the PK of Zalypsis® when administered as a single-agent to patients 
with EFT.

To determine the pharmacodynamic profile by measuring the effect of Zalypsis® on 
the number of Ewing’s sarcoma CTCs at different times of treatment and its 
correlation with the clinical outcome.

To determine the PGx profile. Hypothesis-generating exploratory PGx analyses will 
be conducted to correlate the molecular parameters found in the tumor and blood 
samples of the patients with the clinical results achieved with Zalypsis®.
3.
OVERALL STUDY DESIGN
This is a multicenter, open-label, single-arm, non-comparative, phase II clinical trial 
with single-agent Zalypsis® given as a 1-h i.v. infusion on d1, d8 and d15, q4wk, to 
patients with advanced and/or metastatic EFT who failed to standard chemotherapy.
The primary endpoint of the study is the overall response rate (ORR), defined as the 
percentage of patients with objective response (OR), either complete response (CR) or 
partial response (PR), as defined by the Response Evaluation Criteria in Solid Tumors 
(RECIST) v1.1.
The study will consist of two stages. If in the first stage there are no responders after 
testing the drug on 12 evaluable patients, the trial will be terminated. If there are ≥ 1 
responders the trial will go on to the second stage and 17 additional evaluable patients 
will be accrued. A total of 29 evaluable patients will be studied. If the total number of 
responders is ≤ 2, the drug will be considered as not interesting in the setting of patients 
treated for this disease with this schedule.
Treatment will be administered in the absence of disease progression and/or 
unacceptable toxicity. In case of obtaining a CR, two additional cycles will be 
administered and then the treatment will be stopped.
A disease evaluation will be performed at baseline and every other cycle (±1 week) until 
evidence of disease progression.
Patients will be evaluated using clinical and laboratory assessments before and after 
each treatment cycle. Any treatment-related AEs will be followed-up until the events or 
their sequelae resolve or stabilize at a level acceptable to the Investigator and the 
Sponsor(s). Patients no longer receiving study treatment will be followed up for survival 
(see Section 5.2).


4.
SELECTION OF PATIENTS
4.1.
INCLUSION CRITERIA
In order to be included into the trial, patients have to fulfill all of the following criteria:
1. Voluntary written informed consent, obtained from the patient or his/her 
representative before the beginning of any specific study procedures. 
2. Age ≥ 16 years.
3. Histologically or cytologically confirmed EFT, with recurrent disease.
4. Documented failure to at least one prior chemotherapy regimen for their disease.
5. Radiographic documentation of disease progression at study entry.
6. Eastern Cooperative Oncology Group (ECOG) performance status (PS) score 
≤ 2.
7. Life expectancy ≥ 3 months. 
8. Complete recovery from the effects of drug-related AEs derived from previous 
treatments, excluding alopecia and grade 1 peripheral neuropathy, according to 
the NCI-CTCAE v. 4.0.
9. At least one measurable lesion (“target lesion” according to the RECIST v. 1.1), 
located in a non-irradiated area and adequately measured less than four weeks 
before study entry. Tumors within a previously irradiated field will be 
designated as "non-target" lesions unless progression is clearly documented or 
biopsy proven.
10. Absolute neutrophil count (ANC) ≥ 1.5 x 109/l; platelet count ≥ 100 x 109/l, and 
hemoglobin ≥ 9 g/dl.
11. Adequate renal function: calculated creatinine clearance (using Cockcroft and 
Gault‘s formula) ≥ 30 ml/min.
12. Adequate hepatic function:
a) Total bilirubin  1.5 x upper limit or normality (ULN), unless due to 
Gilbert’s syndrome.
b) Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 3 x 
ULN (≤ 5 x ULN in case of hepatic metastases), and alkaline phosphatase (AP) 
≤ 2.5 x ULN (≤ 5 x ULN in case of extensive bone involvement).
c) Albumin ≥ 25 g/l.
13. Left ventricular ejection fraction (LVEF) within normal limits (LVEF of at least 
50%).
14. Women of childbearing potential must have a negative serum pregnancy test 
before study entry. Both women and men must agree to use a medically 
acceptable method of contraception throughout the treatment period and for 
three months after discontinuation of treatment. Acceptable methods of 
contraception include complete abstinence, intrauterine device (IUD), oral 
contraceptive, subdermal implant and double barrier (condom with a 
contraceptive sponge or contraceptive suppository).


4.2.
EXCLUSION CRITERIA
Patients fulfilling any of the following criteria will not be included into the trial:
1. Prior therapy with Zalypsis®.
2. Pregnant or lactating women or women of childbearing potential not using an 
appropriate contraceptive method.
3. Less than three weeks from prior radiation therapy, biological therapy or 
chemotherapy.
4. Less than six weeks from prior nitrosourea, mitomycin C, high-dose 
chemotherapy or radiotherapy involving the whole pelvis or over 50% of the 
spine, provided that acute effects of radiation treatment have resolved (Appendix
2). Hormonal therapy and palliative radiation therapy (i.e., for control of pain
from bone metastases) must be discontinued before study entry. 
5. Patients with a prior invasive malignancy (except non-melanoma skin cancer and 
in situ cervix carcinoma) who have had any evidence of disease within the last 
five years or whose prior malignancy treatment contraindicates the current 
protocol therapy.
6. Evidence of progressive or symptomatic central nervous system (CNS) 
metastases or leptomeningeal metastases.
7. Other diseases or serious conditions:
a) Increased cardiac risk, as defined by:

Unstable angina or myocardial infarction within 12 months before 
inclusion in the study.

New York Heart Association (NYHA) grade II or greater congestive 
heart failure. 

Symptomatic arrhythmia or any arrhythmia requiring ongoing treatment. 

Abnormal ECG, i.e., patients with the following are excluded: QT 
prolongation - QTc > 480 msec; signs of cardiac enlargement or 
hypertrophy; bundle branch block; partial blocks; signs of ischemia or 
necrosis, and Wolff Parkinson White patterns.

History or presence of valvular heart disease.

Uncontrolled arterial hypertension despite optimal medical therapy.

Previous mediastinal radiotherapy.

Previous treatment with doxorubicin at cumulative doses exceeding 
400 mg/m2.
b) History of significant neurological or psychiatric disorders.
c) Active infection requiring systemic treatment.
d) Significant non-neoplastic liver disease (e.g., cirrhosis).
e) Known hepatitis B virus (HBV) or hepatitis C virus (HCV) infection.
f)
Immunocompromised patients, including those known to be infected with the 
human immunodeficiency virus (HIV).
g) Uncontrolled (i.e., requiring relevant changes in medication within the last 
month or hospital admission within the last three months) endocrine diseases 
(e.g., diabetes mellitus, hypo- or hyperthyroidism, adrenal disorder).
8. Any other major illness that, in the Investigator’s judgment, will substantially 


increase the risk associated with the patient’s participation in the study. The 
Investigator should feel free to consult the Study Coordinator or the Sponsor(s) 
for uncertainty in this regard.
9. Limitation of the patient’s ability to comply with the treatment or to follow-up at 
a participating center. Patients enrolled into this trial must be treated and 
followed at a participating center.
10. Treatment with any investigational product within 30 days prior to inclusion in 
the study.
11. Known hypersensitivity to any component of Zalypsis®.
4.3.
PATIENTS FOR THE PHARMACOGENOMIC SUBSTUDY
4.3.1.
Inclusion Criteria 
1. All patients included in trial PM104-B-003-10 will be eligible.
2. Only those patients who have themselves or their representatives voluntarily 
signed the Informed Consent Form for the PGx study will participate.
4.3.2.
Exclusion Criteria 
1. Patients who do not consent to participate in this substudy (by themselves or 
through their representatives). Refusal to participate in the PGx substudy will not 
affect participation in the main trial PM104-B-003-10.
5.
PLAN OF THE STUDY
5.1.
DURATION OF STUDY (WHOLE POPULATION)
The total duration of the study will be approximately 36 months, including about 24 
months of active enrollment.
Consenting patients will be followed until death, or until the date of study termination, 
whichever occurs first. 
Planned start date (first patient on study): 1Q11.
Planned enrollment period: 24 months.
Planned study termination will occur:

Depending on the development/extension of the trial: 
o
Three months after the last treatment visit of the last evaluable patient if 
the study ends at the first stage.
o
Six months after the last treatment visit of the last evaluable patient, if 
the study proceeds to the second stage.

Or 12 months after the last patient included in the study, if neither of the above 
have occurred.
5.2.
DURATION OF STUDY AND TREATMENT (PER PATIENT)
Patients will receive study treatment as long as it is considered to be in their own benefit 
(see Section 7.2.3). Patients will be evaluated at scheduled visits in up to three study 
periods: 


Pre-treatment: from the signature of informed consent to the first infusion of 
Zalypsis®.
Treatment: from the first infusion of Zalypsis® to the last administration of study 
drug plus 30 days, or onset of subsequent therapy or death, whichever occurs first. 
An end-of-treatment visit will be performed within 30 days after last dose 
administration. 
Follow-up: all patients will be followed up after the end of treatment: 

Patients who discontinue treatment without progression will be followed every 
three months until disease progression, other antitumor therapy or death or 
until the date of study termination, whichever occurs first. 

After disease progression or after other antitumor therapy, patients will be 
followed every three months until death, or until the date of study termination, 
whichever occurs first.
Patients may withdraw their consent at any time; no further study activities will be 
conducted on them.
Patients will be considered to be on-study from the signature of the informed consent to 
the end of follow-up period. Patients will be considered to be on-treatment for the 
duration of their treatment and in the first 30 days following the date of the last study 
drug administration, or until the onset of subsequent therapy or death, whichever occurs 
first. End of treatment is defined as the day of the last study drug dose administration 
plus 30 days, or onset date of subsequent therapy or the date of death, whichever occurs 
first.
5.2.1.
Discontinuations
5.2.1.1. Treatment Discontinuation
The end of treatment occurs when an enrolled patient ceases to receive the study 
medication, regardless of the circumstances. The primary reason for any discontinuation 
will be recorded on the patient’s Case Report Form (CRF). By convention, the date of 
end of treatment will be the day of the last Zalypsis® administration plus 30 days, or the 
date of the onset of subsequent therapy or the date of death, whichever occurs first.
If a patient discontinues treatment, every effort should be made to complete the 
scheduled assessments.
5.2.1.2. Reasons for Treatment Discontinuation
Administration of the study treatment should be discontinued if this is considered to be 
in the best interest of the patient. More specifically, treatment will be discontinued due 
to any of the following reasons:

Disease progression.

Unacceptable toxicity (including any toxicity leading to the need for a third dose 
reduction or severe hypersensitivity reactions, except in the event of obvious 
clinical benefit, in which case the patient could be allowed to remain on 
treatment after having discussed and agreed on the case with the Sponsor(s)).

Patient’s representative refusal and/or non compliance with study requirements.



Intercurrent serious illness. 

Protocol deviation with an effect on the risk/benefit ratio of the clinical trial.

Treatment delay > 2 weeks or impossibility to administer both Day 8 and Day 
15 doses on a given cycle (except in case of clear clinical benefit, with the 
Sponsor(s)’s approval).

Requirement of > 2 dose reductions (except in case of clear clinical benefit, with 
the Sponsor(s)’s approval).

Administrative reasons or Sponsor(s)’s decision.
Regardless of the reason, patients who discontinue the treatment must not be re-treated 
at any time.
Any subsequent therapies for the patients may be provided off-study according to the 
Investigator’s criteria.
5.2.1.3. Study Discontinuation
Study discontinuation occurs when an enrolled patient ceases to participate in the study, 
regardless of the reason. Patients have the right to withdraw consent at any time; if this 
is the case, no further follow-up should be performed.
The date and reason for study discontinuation will be clearly documented on the 
patient’s CRF. 
5.2.2.
Protocol Deviations
A protocol deviation is defined as any departure from what is described in the protocol 
of a clinical trial approved by an Independent Ethics Committee/Institutional Review 
Board (IEC/IRB) and Competent Authorities. Therefore, this applies to deviations 
related to patient inclusion and clinical procedures (e.g., assessments to be conducted or 
parameters to be determined), and also to other procedures described in the protocol that 
concern the Good Clinical Practice (GCP) guidelines or ethical issues (e.g., issues 
related to obtaining the patients’ Informed Consent, data reporting, the responsibilities 
of the Investigator, etc.).
Deviations with no effects on the risk/benefit ratio of the clinical trial (such as minimal 
delays in assessments or visits) will be distinguished from those that might have an 
effect on this risk/benefit ratio, such as:

Deviations that might affect the clinical trial objectives, such as those involving 
the inclusion/exclusion criteria (which could mean that the patient is not eligible 
for the trial) and those having an effect on patient evaluability.

Deviations that might affect the patient’s well-being and/or safety, such as an 
incorrect dosing of the investigational medicinal product (IMP, this is Zalypsis®) 
due to not following dose adjustment specifications or an incorrect preparation 
of the medication.

Deviations related to the following of GCP guidelines as described in the 
protocol and regulations in force, such as deviations when obtaining the 
Informed Consent or not following the terms established for reporting serious 
adverse events, etc.
As a general rule, NO deviations that may have an effect on the risk/benefit ratio of the 
clinical trial will be authorized. All protocol deviations considered particularly relevant 
according to regulations, which are related to ethical issues, fulfillment of GCP 


guidelines and trial procedures, will be notified to the pertinent IEC/IRB and, if 
pertinent, to the competent authorities as established by local regulations.
5.3.
REPLACEMENT OF PATIENTS
Patients must be replaced if they are considered not evaluable for efficacy, i.e., if they 
are withdrawn from the study due to significant clinical deterioration of unknown 
reason, hypersensitivity reactions, patient refusal or unrelated AEs without any tumor 
assessments after the start of study treatment.
5.4.
STUDY TERMINATION (CLINICAL CUTOFF)
Study termination will occur:
Depending on the development/extension of the trial:

Three months after the last treatment visit of the last evaluable patient if the 
study ends during the first stage.

Six months after the last treatment visit of the last evaluable patient, if the 
study proceeds to the second stage.
Or 12 months after the last patient included in the study, if neither of the above have 
occurred.
5.5.
PRE-TREATMENT EVALUATION
During the pre-treatment period, and once the patient or his/her representative has 
signed the Informed Consent Form, the Investigator will confirm the patient’s eligibility 
for the study by conducting the following assessments (Table 5). 
Additional information on the collection and processing of pharmacodynamic and PGx 
samples will be provided as a separate document in the “Guide for Identification, 
Packaging and Shipment of Pharmacodynamic and Pharmacogenomic Samples”.


Table 5. Pre-treatment assessments
PRE-TREATMENT ASSESSMENT
TIME
 Written informed consent.
Prior to any specific study procedures.
 Medical history:
o Date of diagnosis of the primary disease.
o Demographic information (race/ethnicity, 
age).
o Prior treatments (surgery, radiotherapy, 
chemotherapy, immunotherapy), specifying 
the best response and the date of PD.
 Concomitant diseases and treatments. 
 Weight, height and BSA.
 Complete physical examination.
 Vital signs: heart rate, blood pressure and body 
temperature. 
 ECOG PS (Appendix 1).
All within four weeks prior to Day 1 of 
Cycle 1.
Complete physical examination, ECOG PS, 
vital signs, and weight must be repeated on 
Day 1 of Cycle 1, prior to administration of 
the first Zalypsis® infusion.
1. History and clinical 
examination
 ECG*
LVEF* by ECHO.
Within four weeks prior to Day 1 of Cycle 
1; ECG must be repeated on Day 1 of 
Cycle 1, before the administration of the 
first Zalypsis® infusion.
2. Laboratory tests
Hematology: differential WBC (neutrophils, 
lymphocytes), hemoglobin and platelets.
Biochemistry A: liver function tests (ALT, AP, 
AST, LDH, total bilirubin), creatinine, CPK, 
troponin I*, glucose, and serum electrolytes (Na+, 
Cl-, K+, Ca++, Mg++).
Biochemistry B: total protein and albumin.
All within one week prior to Day 1 of 
Cycle 1.
Repeat Hematology and Biochemistry A on 
Day 1 of Cycle 1 prior to treatment with 
Zalypsis® (a window of −2 days allowed), 
if the treatment is administered more than 
one week after the pre-treatment test. 
Repeat Biochemistry B on Day 1 of Cycle 
1 whenever clinically indicated.
3. Calculated 
creatinine clearance
Calculated according to Cockcroft and Gault’s 
formula (Appendix 4).
Within one week prior to Day 1 of Cycle 1.
Repeat on Day 1 of Cycle 1 whenever 
clinically indicated.
4. Pregnancy test, if 
applicable
Within two weeks prior to registration and 
repeat on Day 1 of Cycle 1.
5. Tumor assessment
CT-scan or MRI of all measurable/evaluable sites 
of disease, as per RECIST v. 1.1. 
Documentation of PD is mandatory.
Within four weeks prior to the first dose of 
Zalypsis®.  
6. PK samples
See Section 6.
Blood samples will be collected on Day 1 
of Cycle 1: one before Zalypsis® infusion, 
and one 5 min before the end of infusion.
7. Pharmacodynamic 
and 
pharmacogenomic 
samples, from 
consenting patients
See Section 8.4 and 8.5.
CTCs will be collected on Day 1 of Cycle 1 
before Zalypsis® infusion.  Paraffin-
embedded 
tumor 
tissue 
(obtained 
at 
diagnosis of the tumor) can be collected 
any time after registration.
8. Other tests
When indicated by the clinical and laboratory 
context.
Within four weeks prior to Day 1 of Cycle 
1.
* All troponin I determinations, ECGs and LVEFs will be reviewed by external cardiologists.
ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BSA, body surface area; 
CPK, creatine phosphokinase; CT, computed tomography; CTCs, circulating tumor cell(s); ECG, electrocardiogram; 
ECHO, echocardiography;  ECOG, Eastern Cooperative Oncology Group;; LDH, lactate dehydrogenase; LVEF, left 
ventricular ejection fraction; MRI, magnetic resonance imaging; PD, progressive disease; PK, pharmacokinetics; PS, 
performance status; RECIST, Response Evaluation Criteria In Solid Tumors; WBC, white blood cells.


5.6.
PATIENT REGISTRATION
After ensuring that the patient meets all eligibility criteria and the patient or his/her 
representative has given written informed consent, he/she will be entered into the trial 
by contacting the clinical trial monitor designated by the Sponsor(s) and faxing the 
completed Patient Registration Form (Fast Fact Sheet). The Sponsor(s) will check this 
Registration Form and confirm eligibility. A patient number will be provided to the site 
of enrolment within one working day. This patient number should be used on all future 
documentation and correspondence referred to this patient.
5.7.
EVALUATIONS DURING TREATMENT
The following assessments will be done while the patient is on treatment (Table 6).
A 2-day window is allowed for the different tests and procedures (except where 
otherwise specified).
Table 6. Evaluations during treatment
ASSESSMENT
TIME
 Complete physical examination.
 ECOG PS (Appendix 1). 
Day 1 of Cycle 1 prior to the Zalypsis®
infusion, and then whenever clinically 
indicated.
 Vital signs: heart rate, blood pressure and body 
temperature.
Before any Zalypsis® infusion.
 Weight and BSA.
Repeat on Day 1 of each cycle prior to the 
Zalypsis® infusion, and then whenever 
clinically indicated.
1. Clinical 
examination
 Concomitant disease and treatments. 
Throughout the “on-treatment” period*.
Hematology: differential WBC (neutrophils, 
lymphocytes), hemoglobin and platelets.
Biochemistry A: liver function tests (ALT, AP, 
AST, LDH, total bilirubin), creatinine, CPK, 
troponin I**, glucose, and serum electrolytes (Na+, 
Cl-, K+, Ca++, Mg++) .
Repeat on Days 1, 8 and 15 of each cycle 
prior to Zalypsis® infusion.
If NCI-CTCAE grade  3 occurs, the 
appropriate test(s) should be repeated at 
least every 2-3 days until recovery to 
grade ≤ 1 or baseline.
In case of febrile neutropenia (any grade) 
ANC determination must be repeated 
every day until resolution.
2. Laboratory 
tests
Biochemistry B: total protein and albumin. 
Repeat on Day 1 of each cycle prior to 
Zalypsis® infusion, and then whenever 
clinically indicated.
If NCI-CTCAE grade  3 occurs, the 
appropriate test(s) should be repeated at 
least every 2-3 days until recovery to 
grade ≤ 1 or baseline.
3. Calculated 
creatinine 
clearance
Calculated according to Cockcroft and Gault’s 
formula (Appendix 4).
Repeat on Day 1 of each cycle prior to the 
Zalypsis® infusion, and then whenever 
clinically indicated.
4. ECG**
ECG 
must 
be 
repeated 
prior 
to 
administration of each Zalypsis® infusion 
and if clinically indicated.
5. LVEF**
ECHO.
Repeat every other cycle, with a 1-week 
window, and if clinically indicated.
6. Pregnancy test, 
if applicable
Every four weeks during treatment and 
three months after last Zalypsis®
administration.


ASSESSMENT
TIME
7. AEs
As per NCI-CTCAE v. 4.0.
Throughout the “on-treatment” period*.
Patients withdrawn with Zalypsis®-related 
ongoing AEs must be followed up with 
the appropriate tests until event 
resolution.
8. Tumor 
Assessment
CT-scan or MRI of all evaluable sites of disease, as
per RECIST v. 1.1.
Every other cycle (± 1 week) until PD.
9. PK samples
See Section 6.
Blood samples will be collected on Day 1 
and 8 of Cycle 1, two before Zalypsis®
end of infusion and six therafter, at 30 
min, 2 h, 6 h, 24 h, 48 h and 168 h after 
end of infusion.
10. 
Pharmacodynamic 
samples, from 
consenting 
patients
See Section 8.4.
On Day 1 before each Zalypsis® infusion 
for the first six cycles, and every three 
cycles thereafter.
11. Other tests
When indicated, according to the clinical 
and laboratory context.
*On-treatment period = from first infusion of Zalypsis® to End of treatment.
End of treatment = 30 days after the day of last dose of study drug administration, unless the patient starts a new 
antitumor therapy or dies (whichever occurs first), in which case the date of administration of this new therapy or the 
date of death will be considered the date of end of treatment.
** All troponin I determinations, ECGs and LVEFs will be reviewed by external cardiologists.
ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BSA, body surface area; 
CPK, creatine phosphokinase; CT; computed tomography; ECG, electrocardiogram; ECHO, echocardiogram; ECOG, 
Eastern Cooperative Oncology Group; LDH, lactate dehydrogenase; LVEF, left ventricular ejection fraction; MRI, 
magnetic resonance imaging; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse 
Events; PD, progressive disease; PK, pharmacokinetics; PS, performance status; RECIST, Response Evaluation 
Criteria In Solid Tumors; WBC, white blood cells.
5.8.
EVALUATION AT END OF TREATMENT
The end-of-treatment visit will be scheduled at 30 days after the last Zalypsis® infusion 
(a window of ± 2 days is allowed).
Regardless of the reason for the discontinuation, the same complete workup conducted 
before study entry (except for medical history) will have to be done at this end-of-
treatment visit. This will include the following assessments:

Complete physical examination.

Vital signs.

ECOG PS.

Weight and BSA, if clinically indicated.

Hematology.

Biochemistry A.

Biochemistry B.

Calculated creatinine clearance, if clinically indicated.

Pregnancy test, if applicable.

ECG and LVEF by ECHO. 



Concomitant diseases and treatments.

Safety assessment (AEs).

Tumor assessment.

Other tests (when indicated, according to the clinical and laboratory context).
Adverse events must be reported until 30 days after the last study drug administration. 
All serious adverse events (SAEs) occurring within 30 days of the last study drug 
administration will be reported. Beyond this period of time, only those SAEs suspected 
to be treatment-related will be reported (see Section 9).
The Sponsor(s) will evaluate all safety information that is spontaneously reported by an 
Investigator beyond the time frame specified in the protocol.
5.9.
FOLLOW-UP AFTER END-OF-TREATMENT VISIT
The date and reason of the study discontinuation will be recorded on the patient’s CRF 
(see Section 5.2.1.1).
Patients who discontinue treatment without progression will be followed every three 
months until disease progression, other antitumor therapy or death or until the date of 
study termination, whichever occurs first. After disease progression or after other 
antitumor therapy, patients will be followed every three months until death, or until the 
date of study termination, whichever occurs first.
Patients’ follow-up determinations will include: ECOG PS, vital signs, complete 
physical examination, weight if clinically indicated, hematology, biochemistry A and B, 
concomitant diseases and treatment, calculated creatinine clearance if clinically 
indicated, and tumor assessment.
Follow-up of cardiac assessments (including ECG and LVEF) should be performed at 
three and six months after end of treatment. 
Patients who withdraw consent will not be followed with any study procedures. 
Patients withdrawn with ongoing Zalypsis®-related AEs (including SAEs) will be 
followed-up until the events or their sequelae resolve or stabilize at a level acceptable to 
the Investigator and the Sponsor(s).
Additional parameters will be assessed and/or the frequency of observations will be 
increased at the Investigator’s discretion and according to the nature of the observed 
AEs. In case of death, when available, autopsy data should be provided.
6.
PHARMACOKINETICS
All patients included in this study will be sampled for PK during the first two infusions 
of the first cycle. Blood samples for the measurement of the Zalypsis® plasma 
concentration will be obtained following the sampling schedule detailed in Table 7. 
6.1.
SAMPLE COLLECTION, STORAGE, AND SHIPPING OF THE PK SAMPLES
All PK sample collection dates and times will be recorded on the PK sheet.
In the treatment infusions with plasma sampling for PK, the infusion rate will be 
established so as to ensure that the total dose is infused in 1 hour. The drug will be 
infused at a constant rate throughout the 1-hour period. In order to obtain reliable PK 
information, the infusion rate should not be modified once it has begun. If a variation 
in the infusion time occurred, it must be recorded on the CRF and PK sheet, stating 


clearly the time of the beginning and the end of the infusion. The infusion rate should 
not be changed to maintain the intended duration of infusion. It would be enough to 
record the actual duration on the CRF and on the PK sampling sheet for those cycles in 
which PK sampling is performed.
Blood samples for PK analysis will be obtained through a peripheral vein located in the 
contralateral side to the study drug infusion. No sample (even the last one) must ever 
be collected from the catheter employed to administer Zalypsis®. If a blood sample 
is obtained from another catheter, the first milliliter (ml) of blood will be discarded to 
avoid dilution of the sample with the solution used to keep it permeable.
Five-ml blood samples will be collected in sodium heparin tubes. Sample tubes will be 
gently inverted several times to ensure adequate mixing and immediately centrifuged at 
2500 x g for 15 min at +4°C to separate the plasma. If immediate centrifugation is not 
possible, the tubes containing the blood samples must be placed in an ice bath at 0-4°C 
for a maximum of 30 minutes. After centrifugation, the plasma will be transferred to the 
provided polypropylene tubes (one per sample) and stored at -20ºC until shipped to the 
analysis laboratory. The cell pellet should be discarded. Blood and plasma tubes will be 
provided by the Sponsor(s).
Blood samples will be collected on Day 1 and 8 of Cycle 1, at the time points shown in 
Table 7. 
Table 7. Plasma pharmacokinetic sampling schedule
Sample number
Optimal time point
Adequate time frame
1
Pre-SOI
Pre-SOI
2
5 min before EOI
5-1 min before EOI
3
30 min after EOI
30 min-1 h after EOI
4
2 h after EOI
1.5-3 h after EOI
5
6 h after EOI
6-8 h after EOI
6
24 h after EOI
20-28 h after EOI
7
48 h after EOI
40-72 h after EOI
  8*
168 h after EOI
120-168 h after EOI
*Sample number 8 has to be taken BEFORE the start of the next infusion.
SOI, start of infusion; EOI, end of infusion.
The accurate recording of actual dosing and sampling times is much more important than 
the strict adherence to the scheduled times.
Once all PK samples from a patient have been collected, they should be shipped to the 
central laboratory for PK analyses as soon as possible, ideally the next shipping day. The
time span between the moment when the last PK sample for a patient has been collected 
and the shipment to the central laboratory of all samples from this patient should not 
exceed two months.
Samples will be sent for analysis in the boxes provided for this purpose, filled with dry ice, 
by the study courier service to the following address:
Mr. Alan Gibbs
Icon Development Solutions
Manchester Bioanalytical Laboratory
Parkway One; Parkway Business Centre
300 Princess Road
Manchester
M14 7QU, United Kingdom


Samples will be identified with the following data: study reference, patient number, sample 
number, date and time of collection. At all times, the confidentiality of patient’s data will 
be maintained.
Samples will be destroyed following the appropriate laboratory procedures, after the 
approval of the final analytical study report by the Sponsor(s).
A manual of instructions for sample collection, labeling, storage, and shipment will be 
provided (Instruction Manual for the Collection, Labeling, Storage and Shipment of 
Pharmacokinetic Samples).
7.
TREATMENT
7.1.
DESCRIPTION OF TREATMENT
Zalypsis® will be administered as a 1-hour i.v. infusion on d1, 8, 15, q4wk, to patients 
with advanced and/or metastatic Ewing’s sarcoma previously treated with at least one 
prior line of chemotherapy.
For instructions regarding drug inventory, handling, reconstitution, dilution, storage, 
accountability and disposal, please refer to the Preparation Guide for Zalypsis® and the 
Zalypsis® IB, both provided as separate documents.
7.2.
ADMINISTRATION OF STUDY MEDICATION
7.2.1.
Formulation
PM001004 (Zalypsis®, 2.5 mg/vial) is provided as a powder for concentrate for solution 
for infusion in only one strength of 2.5 mg. 
7.2.2.
Dose Schedule
PM001004 (Zalypsis®) will be administered at a dose of 2 mg/m2. A treatment cycle 
consists of the drug administration on Days 1, 8 and 15, plus one week of follow-up 
(study evaluations should be completed during each cycle prior to subsequent Zalypsis®
infusion). Treatment cycles will be repeated every four weeks. 
7.2.3.
Criteria for Treatment Continuation
Patients will remain on treatment in the absence of confirmed or unacceptable toxicity 
that is not resolved after applying the appropriate dose reductions.
In order to be re-treated on Day 8 and Day 15 of the first cycle and on subsequent 
infusions, the patients will have to fulfill the following criteria:
a) Platelet count  75 x 109/l, hemoglobin  9 g/dl and ANC  1.0 x 109/l.
b) AP  2.5 x upper limit of normality (ULN) (≤ 5 x ULN in case of extensive bone 
involvement).
c) ALT and AST ≤ 3 x ULN (≤ 5 x ULN in case of hepatic metastases).
d) Renal function: patients with calculated creatinine clearance (using Cockcroft 
and Gault's formula)  30 ml/min.
e) Total bilirubin  1.5 x upper limit or normality (ULN), unless due to Gilbert’s 
syndrome.
f)
Albumin ≥ 2.5 g/dl.
g) Other non-hematological toxicities grade  1 (grade 2 in case of asthenia). 


If these criteria are not met on Day 8, 15 or on subsequent infusions, study drug 
administration should be skipped and criteria reevaluated weekly. Treatment 
administration will be resumed upon recovery of these parameters, according to these 
same criteria.
If a patient has to skip two doses (at Day 8 and Day 15) or has a delay > 2 weeks from 
the theorical day of re-treatment, the patient should be discontinued from treatment, 
except in the event of obvious clinical benefit, in which case the patient may be allowed 
to remain on treatment only after having discussed and agreed on the case with the 
Sponsor(s), and upon subsequent recovery of all parameters according to the 
aforementioned criteria. 
7.2.4.
Dose Reduction
Zalypsis® dose reductions will take place based on the worst treatment-related toxicity 
found since the last dose administration. The criteria for dose reduction are:

Grade 4 neutropenia that lasts > 5 days.

Febrile neutropenia.

Grade 4 thrombocytopenia.

Grade ≥ 3 transaminase increase that lasts > 7 days.

Any other grade 3-4 adverse event non-hematologic toxicity except nausea, 
vomiting and diarrhea (unless grade 3 or 4 nausea, vomiting and diarrhea 
persists despite use of adequate medication).

In the event of a patient requiring dose reduction, the new Zalypsis® dose level 
is described in Table 8.
Table 8. Dose modification according to toxicity
Dose level
Zalypsis® (mg/m2)
1
2.0
-1
1.6
-2
1.3
Following the resolution of any of the aforementioned toxicities, patients may continue 
treatment at one dose level below. If treatment-related toxicities reappeared in the 
following drug administrations, a second dose reduction to a level below is allowed. 
No more than two dose reductions per patient will be allowed during the study. Patients 
requiring more than two dose reductions should discontinue the treatment, except in the 
event of obvious clinical benefit, in which case the patient could be allowed to remain 
on treatment after having discussed and agreed on the case with the Sponsor(s).
No dose escalations will be allowed in this study.


7.3.
CONCOMITANT MEDICATION
All tumor-specific prior chemotherapy, radiation therapy and all relevant information 
must be recorded on the patient's CRF.
In addition, reasonable efforts must be made to determine all treatments received by the 
patient during administration of the study treatment. This information must be 
documented in the concomitant therapy section of the CRF.
7.3.1.
Prophylactic Antiemetic Treatment
According to the American Society of Clinical Oncology (ASCO) guidelines for drugs 
with moderate emetic risk (36), before the infusion of Zalypsis® the patients will receive 
prophylactic treatment for emesis for adult patients consisting of dexamethasone 8 mg 
i.v. and 5-HT3 antagonists (ondansetron 8 mg i.v. or granisetron 1 mg i.v. or tropisetron 
5 mg i.v.).
If necessary, one or both of the following may also apply:

Adding 10 mg of metoclopramide orally every eight hours.

Extending the duration of treatment with 5-HT3 antagonists and/or 
dexamethasone.
7.3.2.
Concomitant Medication Permitted

Therapies for preexisting and treatment-emergent medical conditions.

Prophylactic antiemetic treatment (described in Section 7.3.1).

Secondary prophylaxis with colony-stimulating factors such as granulocyte (G-
CSF) or granulocyte-macrophage (GM-CSF) colony-stimulating factors are 
allowed according to the ASCO guidelines (37).

Erythropoietin therapy or derivatives (38).

Bisphosphonates.

In case of diarrhea (39) and pruritus, appropriate treatment will be permitted, 
including topical and systemic corticosteroids and antibiotics. 

Palliative local radiation may be applied. The irradiated lesion will then not be 
considered an area of measurable/evaluable disease.
7.3.3.
Concomitant Medication Prohibited

Concomitant administration of any other antineoplastic therapy.

Investigational agents.

Immunosuppressive therapies, including systemic corticosteroids, unless used as 
therapy for preexisting and treatment-emergent medical conditions (e.g., emesis, 
rash, anorexia).

Primary prophylaxis (i.e., before the intended preventable events occurs) with 
colony-stimulating factors such as granulocyte (G-CSF) or granulocyte-
macrophage (GM-CSF) colony-stimulating factors. Their use as secondary 
prophylaxis is permitted according to the ASCO guidelines (37). However, dose 
reduction will be applied if indicated, regardless of the use of secondary 
prophylaxis with colony-stimulating factors.


7.4.
PACKAGING AND LABELING
7.4.1.
Zalypsis®
PM00104 (Zalypsis®) is provided as a sterile lyophilized powder for concentrate for 
solution for infusion. Each vial will contain 2.5 mg of PM00104.
The labels will contain information that will meet applicable regulatory requirements.
7.5.
DRUG ACCOUNTABILITY
Proper drug accountability will be done by the clinical trial monitor. Each study site will 
keep records to allow a comparison of quantities of drug received and used at each site. 
The Investigator at each study site will be the person ultimately responsible for drug 
accountability at the site.   
All unused drug supplied by the Sponsor(s) will be properly destroyed at the study site. 
Documentation of this procedure must be provided to the clinical trial monitor. If the 
Sponsor(s) agree, unused drug supplies may be returned to the drug repository.
7.6.
TREATMENT COMPLIANCE
The Investigator is responsible for supervising compliance with the instructions 
described in this study protocol.
8.
STUDY EVALUATIONS
8.1.
EFFICACY
To be evaluable for efficacy, patients must have received at least four of the six 
infusions in the first two cycles (e.g., two infusions in each cycle, or three infusions in 
Cycle 1 and one infusion in Cycle 2), and have at least one disease measurement 
recorded not less than six weeks after treatment onset. 
In addition, any eligible patient who receives at least two of the three infusions in one 
treatment cycle and presents disease progression or dies due to progressive disease (PD) 
prior to response evaluation will be considered evaluable for the main endpoint (ORR) 
and will be categorized as an “early progression”.
Patients withdrawn due to toxicity without any tumor assessment after the start of study 
treatment will be considered as “treatment failures” and will not be replaced.
Patients withdrawn due to significant clinical deterioration of unknown reason, or due to 
hypersensitivity reactions or unrelated AEs, and patients who refuse to continue on 
study for any reason without any tumor assessment after the start of study treatment will 
be considered not evaluable for efficacy and will be replaced.
Assessment of efficacy will be done using the RECIST v1.1 and will be essentially 
based on a set of measurable lesions identified at baseline as target lesions and followed 
until disease progression. A comprehensive workup will be performed at baseline and 
every other cycle (± 1 week) until evidence of PD. The same procedure will be used to 
evaluate each identified lesion both at baseline and throughout the treatment period.
In case of detection of an OR, either CR or PR, a confirmation assessment has to be 
performed a minimum of four weeks after the first documentation of the response.


Additionally, detection of the presence of circulating CTCs by retrotranscriptase 
polymerase chain reaction (RT-PCR) analysis of EWS-FLI1 and EWS-ERG 
translocations in the blood of patients will be performed.
The time-to-event parameters that will be analyzed are detailed in Section 10.2.2.
A reference to the RECIST v. 1.1 may be found in Appendix 3. 
8.2.
SAFETY
Patients will be evaluable for safety if they have received at least one total or partial 
infusion of Zalypsis®. 
Safety will be evaluated using clinical examinations, which will comprise vital signs 
analysis, clinical assessment of AEs, changes in laboratory parameters (hematological 
and biochemical, including liver and cardiac function tests), and any other analyses that 
may be considered necessary.
All AEs will be classified according to the NCI-CTCAE, v. 4.0 and will be coded using 
the Medical Dictionary for Regulatory Activities (MedDRA) v. 11.0.
8.3.
CENTRAL CARDIOLOGICAL REVIEW
Because of safety reasons, an independent cardiologist will undertake a review of all the 
ECGs and ECHOs performed to the patients during the course of the study, in order to 
improve the consistency in the evaluation criteria applied. 
Originals or copies of all ECGs, ECHOs, and laboratory records of troponin I, need to 
be provided by the Investigator to the Independent Reviewer on ongoing basis. Other 
patient clinical or laboratory records may need to be provided by the Investigator upon 
request from the Independent Reviewer. 
In the event of disagreement between the Investigator's and the centralized assessment, 
discussion will take place between the two parties in order to reach a consensus. 
However, if it is not forthcoming, it is the assessment of the independent reviewer, 
which will be retained in the report of the Expert Review, and in intermediate and final 
reports.
8.4.
PHARMACODYNAMIC SUBSTUDY
A secondary endpoint of the study is the enumeration of Ewing’s sarcoma CTCs, not 
only as a prognostic factor but also as a pharmacodynamic marker of response to 
Zalypsis® treatment. A unique characteristic of the Ewing’s sarcoma tumor cells is 
having a specific chromosomal translocation that can be determined with high 
specificity in blood samples by quantitative real-time retrotranscriptase polymerase 
chain reaction (qRT-PCR), even in the presence of an excess of normal blood cells of 
several orders of magnitude. Thus, the chromosomal translocation will be initially 
identified by FISH in tumor samples and latter assayed in the blood samples.
This pharmacodynamic substudy will analyze the presence of Ewing’s sarcoma cells in 
the blood of patients before Zalypsis® infusion in each cycle of treatment. It is expected 
that most patients in the advanced metastatic setting will be positive for the presence of 
Ewing’s sarcoma cells in their blood, as detected by qRT-PCR. It seems reasonable that 
an effective treatment with Zalypsis® would lead to the reduction of CTCs as it has been 
described for other first-line treatments in this tumor type (41). Similarly, the 
reappearance of the CTCs could be used as an early marker of PD.
To perform this pharmacodynamic substudy it is required the collection of 8 ml of 


peripheral blood in Vacutainer® CPTTM tubes from the patient immediately before the 
infusion of Zalypsis® in each treatment cycle. The blood sample has to be immediately 
centrifuged to separate plasma and white blood cells and stored frozen until analysis.
The number of Ewing’s sarcoma CTCs at each time point will be determined by qRT-
PCR and correlated with the patient’s outcome.
For detailed information on the collection and shipping of CTCs samples please refer to 
the Guide for Identification, Packaging and Shipment of Pharmacodynamic and 
Pharmacogenomic samples.
8.5.
PHARMACOGENOMIC SUBSTUDY
The identification of PGx biomarkers of response to Zalypsis® is a secondary endpoint 
of this study to be evaluated in a PGx substudy. The aim of this PGx substudy is to 
identify and validate molecular markers whose expression may be associated with the 
clinical outcome of patients treated with Zalypsis®. These molecular markers will allow 
the identification of those patients who would benefit from the treatment with 
Zalypsis®, improving the health care by an individualized medicine.
The experimental data points out to Zalypsis® as a DNA binder that produces DSBs in 
the DNA that are repaired by homologous recombination repair. In addition, Zalypsis®
has also been involved in transcription, interfering both with transcription initiation and 
elongation (PharmaMar internal report). Then, it seems rational to develop studies to 
conduct correlations between the outcome of tumor/patient exposed to Zalypsis® and 
genes/proteins determinant in the efficiency/deficiency of the DNA repair pathways and 
transcription. The ultimate goal is the characterization of such patients that may be 
prone to respond to Zalypsis® in order to implement a customized therapy in the future.
Several genes, which have been identified as related to the response to Zalypsis® in 
mammalian cell lines and in yeast models, will be analyzed as potential markers of 
response in patients treated with Zalypsis®. Initially PDGFRa and PARP genes will be 
analyzed. Additonal genes related to Zalypsis® MoA could be selected for analysis later 
on. The expression of those genes in patient paraffin-embedded tumor tissue will be 
determined, analyzing mRNA levels by qRT-PCR, and protein expression by 
immunohistochemistry (using specific antibodies).
The ideal sample for gene mRNA expression analysis is the paraffin-embedded tumor 
tissue obtained at the diagnosis of the disease, because it is frequently conserved in the 
pathology archives. Tumor tissue slices in polyethylene naphthalate (PEN) membrane 
slides are microdissected to isolate tumoral cells and total RNA is extracted and the 
mRNA expression of selected genes is performed by qRT-PCR using gene specific 
primers.
In addition, the same tissue block samples used in the mRNA expression analysis will 
be studied at the protein level by tissue microarrays. To do so, cylinders from the 
paraffin-embedded tissue blocks will be cut and arrayed in a new paraffin block to allow 
the simultaneous determination of the expression of proteins by immunohistochemistry 
using specific antibodies. Thus, the expression of several proteins and its histological 
localization can be analyzed and correlated with the clinical outcomes of the 
corresponding patients, in a technical setting more familiar for the Pathology 
Departments. 
PharmaMar will provide a Guide for Identification, Packaging and Shipment of 
Pharmacodynamic and Pharmacogenomic Samples detailing the procedures to follow 


for sample collection, labeling and shipment. PharmaMar is responsible for selecting 
reference laboratories with demonstrated skills to perform the tests planned.
A positive consequence in the upcoming development of Zalypsis® is to be expected if 
we are able to identify a correlation between the expression of RNA/protein in the 
tumor tissue and the patient clinical outcomes after Zalypsis® treatment, since this 
active drug could be then given in a customized way to advanced resistant cancer 
patients, thus maximizing the rate of clinical benefit.
9.
ADVERSE EVENTS REPORTING
9.1.
DEFINITIONS
9.1.1.
Adverse Event
An adverse event (AE) is any untoward medical occurrence in a patient or a clinical 
investigation patient administered a pharmaceutical product which does not necessarily 
have a causal relationship with this treatment. 
An AE can therefore be any unfavorable and unintended sign (e.g., an abnormal 
laboratory finding), or a disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product.
Any event involving adverse drug reactions, illnesses with onset during the study or 
exacerbations of pre-existing illnesses should be recorded, including but not limited to 
clinically significant changes in physical examination findings and abnormal objective 
test findings (e.g., ECG). The criteria for determining whether an abnormal objective 
test finding should be reported as an AE are as follows:

The test result is associated with clinically significant symptoms, and/or,

The test result leads to a change in the study dosing or discontinuation from the 
clinical trial, significant additional concomitant drug treatment or other therapy, 
and/or,

The test result leads to any of the outcomes included in the definition of a SAE 
and/or,

The test result is considered to be an AE by the Investigator. 
Tumor progression or appearance of new tumor lesions MUST NOT be reported as an 
AE.
9.1.2.
Serious Adverse Event
A Serious Adverse Event (SAE) is any adverse experience occurring at any dose that:

Results in death (is fatal),

Is life-threatening,

Requires or prolongs inpatient hospitalization,

Results in persistent or significant disability or incapacity,

Is a congenital anomaly or birth defect, 

Is medically significant, or

Is any suspected transmission of an infectious agent via a medicinal product. 


Medical and scientific judgment should be exercised in deciding whether expedited 
reporting is appropriate in other situations such an important medical event that may not 
be immediately life-threatening or result in hospitalization but may jeopardize the 
patient or may require intervention to prevent one of the outcomes listed in the above 
definition.
Tumor progression or appearance of new tumor lesions MUST NOT be reported as a 
SAE.
9.1.2.1. Death
Death as such is the outcome of a SAE and should not be used as the SAE term 
itself. Instead, the cause of death should be recorded as the SAE term. When available, 
the autopsy report will be provided to the Sponsor(s).
9.1.2.2. Life-threatening Event
Any event in which the patient 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.
9.1.2.3. Hospitalization or Prolongation of Hospitalization 
Any AE requiring hospitalization (or prolongation of hospitalization) that occurs or 
worsens during the course of a patient’s participation in a clinical trial must be reported 
as a SAE unless exempted from SAE reporting (see Section 9.2.2). Prolongation of 
hospitalization is defined as any extension of an inpatient hospitalization beyond the 
stay anticipated/required for the initial admission, as determined by the Investigator or 
by the treating physician.
Hospitalizations that do not meet criteria for SAE reporting are: 
a.
Reasons described in protocol [e.g., investigational medicinal product (IMP) 
administration, protocol-required intervention/investigations, etc]. However, events 
requiring hospitalizations or prolongation of hospitalization as a result of a 
complication of therapy administration or clinical trial procedures will be reported 
as SAEs.
b. Hospitalization or prolonged hospitalization for technical, practical or social 
reasons, in absence of an AE.
c.
Pre-planned hospitalizations: any pre-planned surgery or procedure must be 
documented in the source documentation. Only if the pre-planned surgery needs to 
be performed earlier due to a worsening of the condition, should this event 
(worsened condition) be reported as a SAE. 
Other situations that MUST NOT be considered as hospitalizations are the following:
d. An emergency visit due to an accident where the patient is treated and discharged.
e.
When the patient is held 24 hours for observation and finally is not admitted.
f.
Planned treatments at sites not associated to a hospital and generally considered as 
minor surgical procedures (e.g., laser eye surgery, arthroscopy, etc).
9.1.3.
Unlisted/Unexpected Adverse Event
An AE, the nature or severity of which is not consistent with the applicable reference 
safety information. The Sponsor(s) will use as the reference safety information for the 
most updated IB for the studied IMP.


9.1.4.
Assessment of Causal Relationship to the Study Drug/IMP
The Investigator must provide an assessment of causal relationship of the clinical trial 
IMPs to each SAE according to the following scale:
Y
There is a reasonable possibility that the IMP caused the SAE.
N
There is no reasonable possibility that the IMP caused the SAE and other 
causes are more probable.
Uk.
Only to be used in special situations where the Investigator has 
insufficient information (i.e., the patient was not seen at his/her centre) if 
none of the above can be used.
9.1.5.
Adverse Events Related to Study Drug (IMP)
An AE is considered related to a study drug/IMP if the Investigator’s assessment of 
causal relationship to the IMP is “Y” (yes). 
The Investigator will assess the causal relationship of each of the IMP to the SAE.
The Sponsor(s) will also consider related to the study drug/IMP those events for which 
the Investigator assesses the causal relationship with the IMP as “Uk” when it cannot 
rule out a role of the IMP in the event. See Section 9.1.4 for causality scale.
9.1.6.
Expedited Reporting
The Sponsor(s) is responsible for the appropriate expedited reporting of SAEs to the 
Regulatory Authorities. The Sponsor(s) will also report all SAEs that are unlisted and 
related to the study drug (IMP), to the Investigators and to the IECs/IRBs according to 
the current legislation unless otherwise required and documented by the IECs/IRB.
9.2.
PROCEDURES
9.2.1.
Reporting Adverse Events
The Sponsor(s) will collect AEs until 30 days after administration of the last dose of 
study drug/IMP or until the start of a new antitumor therapy, whichever occurs first. All 
AEs suspected to be related to the study drug/IMP must be followed after the time of 
therapy discontinuation until the event or its sequelae resolve or stabilize at an 
acceptable level to the Investigator and the Sponsor(s).
All AEs must be recorded using medical terminology in the source document and the 
CRF. Whenever possible, the Investigator will record the main diagnosis instead of the 
signs and symptoms normally included in the diagnoses. 
Investigators must assess severity (grade) of the event following the NCI-CTCAE v. 4.0 
and assign relationship to the study drug/IMP; pursue and obtain information adequate 
both to determine the outcome and to assess whether it meets the criteria for 
classification as a SAE requiring immediate notification to the Sponsor(s) or its 
designated representative. The Investigator must provide any relevant information as 
requested by the Sponsor(s) in addition to that on the CRF.
Disease progression should not be reported as an adverse event and will only be 
reported in the applicable CRF page.
Abnormal laboratory tests occurring during the study should be only recorded in the AE 
section of the CRF if the disorder:



is associated with clinically significant symptoms, and/or 

leads to a change in study dosing or discontinuation from the study, significant 
additional concomitant drug treatment or other therapy, and/or 

leads to any of the outcomes included in the definition of a SAE. 
Otherwise laboratory results should be reported in the corresponding section of the CRF 
(Biochemistry or Hematology).
9.2.2.
Reporting Serious Adverse Events
The Sponsor(s) will collect SAEs from the signing of the Informed Consent Form. If the 
patient is definitively included in the study, this information will also be recorded in the 
AE section of the CRF. 
SAEs will be collected until 30 days after administration of the last dose of study 
drug/IMP or until the start of a new antitumor therapy, whichever occurs first. Beyond 
this period of time, only those SAEs suspected to be related to the IMP/s need to be 
reported. Nonetheless, the Sponsor(s) will evaluate any safety information that is 
spontaneously reported by an Investigator beyond the time frame specified in the 
protocol.
All SAEs (as defined above) regardless of relationship to the study drug/IMP must be 
reported immediately and always within 24 hours to the PharmaMar Pharmacovigilance 
function by fax (+34 91 846 6004) or telephone (+34 91 846 6054). Out of office hours 
(GMT), assistance on SAE reporting can be obtained by calling the Pharmacovigilance 
Service at +34 91 823 4749.
The preferred reporting method is by faxing the completed SAE form. An initial report 
by telephone must be followed by a completed “Serious Adverse Event Form” from the 
investigational staff within one working day.
All SAEs suspected to be related to the IMP/s must be followed until the event or its 
sequelae resolves or stabilizes at an acceptable level by the Investigator and the clinical 
monitor or his/her designated representative.
Exemptions from SAE REPORTING:  Events of “disease progression” even if they 
fulfill any seriousness criterion (i.e. fatal, requiring hospitalization, etc) are exempted 
from SAE reporting and will only be reported in the applicable CRF page.
9.2.3.
Reporting Pregnancy Cases Occurring During the Clinical Trial
National regulations require that clinical trial Sponsor(s) collect information on 
pregnancies occurring during clinical trials, in which exposure to the IMPs at any time 
during pregnancy, via either maternal or paternal exposure, is suspected.
Therefore, pregnancy and suspected pregnancy (including a positive pregnancy test 
regardless of age or disease state) of a female patient or the female partner of a male 
patient occurring while the patient is on study drug, or within 30 days of the patient’s 
discontinuation visit, are considered immediately reportable events.
The Investigator will report the following events immediately and always within 24 
hours from first knowledge:

Any occurrence of a pregnancy where any kind of exposure to the IMP/s is 
suspected.

Possible exposure of a pregnant woman (this could involve a partner of a male 
patient or a pregnant female who came in contact with the clinical trial IMP/s).



All reports of elevated/ questionable or indeterminate beta human chorionic 
gonadotrophins (βhCGs).
Immediately after detecting a case of suspected pregnancy in a female clinical trial 
patient, the decision on her continued participation in the clinical trial will be jointly 
taken by the trial patient, the Investigator and the Sponsor(s), with the patient’s best 
interest in mind. A decision to continue the pregnancy will require immediate 
withdrawal from the trial. 
Any pregnancy, suspected pregnancy, or positive pregnancy test must be reported to 
PharmaMar Pharmacovigilance immediately by facsimile using the Pregnancy Report 
form. In the case of pregnancy of the female partner of a trial Patient the Investigator 
will obtain her informed consent to provide the information by using the applicable 
form provided by the Sponsor(s) who will also advise the Investigator in these 
situations.
The Investigator will follow the pregnancy until its outcome, and must notify 
PharmaMar Pharmacovigilance the outcome of the pregnancy within 24 hours of first 
knowledge as a follow-up to the initial report.
For any event during the pregnancy, which meets a seriousness criterion (including fetal 
or neonatal death or congenital anomaly), the Investigator will also follow the 
procedures for reporting SAEs (complete and send the SAE form to PharmaMar 
Pharmacovigilance by facsimile within 24 hours of the Investigator’s knowledge of the 
event).
All neonatal deaths that occur within 30 days of birth should be reported, without regard 
to causality, as SAEs. In addition, any infant death at any time thereafter that the 
Investigator suspects is related to the exposure to the study drug/IMP should also be 
reported to PharmaMar Pharmacovigilance by facsimile within 24 hours of the 
Investigators’ knowledge of the event.
9.2.4.
Periodic Safety Review of Clinical Data
Although periodic safety review of clinical data will be performed, no formal Data 
Safety and Monitoring Board (DSMB) has been appointed for this exploratory, small 
sample-size trial. Adverse events will be monitored by the Investigators and by the 
study team at PharmaMar. 
The personnel in charge of this process are defined on the section “Study Contacts” of 
the protocol. A medical oncologist will review the safety data of this trial on an ongoing 
basis. 
SAEs will be collected, assessed and reported as per the applicable Regulations by the 
Pharmacovigilance Department / Regulatory Affairs Department.
In the case of an accumulation of unexpected SAEs, stopping guidelines have been 
established in the protocol (see Section 5.2). 
As per the applicable regulations, the Sponsor(s) will report to IECs/IRBs, investigators 
and Regulatory Authorities:
• expeditedly: all serious, related, unexpected AEs or critical safety findings from 
this and any other clinical trial with the same study drug/ IMP, and 
• periodically: within an Annual Report: all the relevant safety information 
generated in all clinical trials with the study drug/ IMP. 


Non-serious AEs will be assessed during monitoring visits by the monitor who will 
discuss them with investigators. Any protocol deviation will also be discussed with the 
investigator during monitoring visits. Safety review will be performed at PharmaMar 
once CRFs have been monitored, collected and shipped to the Sponsor(s).
10.
STATISTICAL METHODS
The statistical analysis will be done by the Sponsor(s) or under the Sponsor(s)’s 
authority.
10.1.
SAMPLE SIZE
The optimum two-stage design to test the null hypothesis that the overall response rate 
(ORR) is ≤ 3% versus the alternative that ORR ≥ 20% was selected. After testing the 
drug on 12 patients in the first stage, the trial will be terminated if there is not any 
responder. If the trial goes on to the second stage, a total of 29 patients will be studied, 
12 from the first stage and 17 from the second stage. If the total number responding is 
less than or equal to 2 (two patients), the drug will be considered not interesting in the 
setting of patients treated with this disease and with this schedule. This design has an 
expected sample size of 17.20 and a probability of early termination of 0.69. If the drug 
is actually not effective, there is a 0.05 probability of concluding that it is (type I error). 
If the drug is actually effective, there is a 0.1 probability of concluding that it is not 
(type II error).
10.2.
ENDPOINTS
10.2.1.
Primary Endpoints
Overall response rate (ORR), defined as the percentage of patients with confirmed 
O), either CR or PR, according to the RECIST v1.1.
10.2.2.
Secondary Endpoints
Efficacy:
Duration of response (DOR), defined as the time between the date when the 
response criteria (PR or CR, whichever is first reached) are fulfilled and the first 
date when disease progression, recurrence or death is objectively documented 
(taking the smallest measurements documented since the treatment started as 
reference for PD).
Progression-free survival (PFS), defined as the time from the first day of study 
treatment to the day of negative assessment (progression or death), start of 
subsequent antitumor therapy, or last tumor evaluation. PFS at 3 and PFS at 6 
months are defined as the Kaplan-Meier estimates of PFS at these time points.
Overall survival (OS), defined as the time from the first day of treatment to the date 
of death (or the last day when the patient is known to be alive). Survival will be 
followed for up to six months after the last treatment visit of the last patient or 12 
months after the last patient is included, whichever occurs first. OS at 12 months is 
defined as the Kaplan-Meier estimates of OS at this time point.
Safety:
Clinical examinations. 
Vital signs analysis. 


Clinical assessment of AEs and SAEs. 
Changes in laboratory parameters: hematological and biochemical (including liver 
and cardiac function tests).
Reasons for study discontinuations, dose delays, omissions, and reductions.
Any other analyses that may be considered necessary to characterize the safety 
profile of Zalypsis® in patients with advanced and/or metastatic EFT.
Others:
Pharmacokinetic profile.
Pharmacogenomic profile.
Pharmacodynamic profile.
10.3.
STATISTICAL ANALYSIS
Continuous variables will be tabulated and presented with summary statistics (i.e., 
mean, standard deviation, median and range). Categorical variables will be summarized 
in frequency tables by means of counts and percentages.
10.3.1.
Efficacy Analysis
All patients who have received at least four of the six infusions in the first two cycles 
(i.e., two infusions in each cycle, or three infusions in Cycle 1 and one infusion in Cycle 
2), and at least one disease measurement recorded not less than six weeks after 
treatment onset.
In addition, any eligible patient who receives at least two of the three infusions in one 
treatment cycle and experience disease progression or die due to progressive disease 
(PD) prior to response evaluation will be considered evaluable for the main endpoint 
(ORR) and will be categorized as an “early progression”.
Patients withdrawn due to toxicity without any tumor assessment after the start of study 
treatment will be considered as “treatment failures” and will not be replaced.
Patients withdrawn due to significant clinical deterioration of unknown reason, 
hypersensitivity reactions, who refuse to continue on study for any reason or unrelated 
AEs without any tumor assessment after the start of study treatment, will be considered 
not evaluable for efficacy and will have to be replaced. 
Assessment of efficacy will be done using the RECIST v. 1.1 and will be essentially 
based on a set of measurable lesions identified at baseline as target lesions and followed 
until disease progression. A disease evaluation will be performed at baseline and every 
other cycle (± 1 week) until evidence of PD. The same procedure will be used to 
evaluate each identified lesion both at baseline and throughout the treatment period.
In case of detection of an OR, either CR or PR, a confirmation assessment has to be 
performed a minimum of four weeks after the first documentation of the response.
Binomial estimates with exact 95% confidence intervals will be calculated for the 
analysis of the main endpoint (ORR).
Time-to-event endpoints (DOR, PFS and OS, PFS at 3 and 6 months and OS rates at 12 
months) will be analyzed according to the Kaplan-Meier method.


10.3.2.
Safety Analysis
Patients will be evaluable for safety if they have received at least one total or partial 
infusion of Zalypsis®. 
Safety will be evaluated using clinical examinations, which will comprise vital signs 
analysis, clinical assessment of AEs, changes in laboratory parameters (hematological 
and biochemical, including liver and cardiac function tests, deaths, reason for study 
discontinuations, dose delays, omissions and reductions, and any other analyses that 
may be considered necessary to characterize the safety profile of Zalypsis® in 
unresectable locally advanced and/or metastatic EFT. Cardiac tests as troponin I, ECG 
and LVEF measurements will be analyzed descriptively.
All AEs will be classified according to the NCI-CTCAE, v 4.0, and will be coded using 
the MedDRA, v. 11.0.
10.3.3.
Pharmacokinetic Analysis
Pharmacokinetic parameters will be calculated using population methods, after pooling 
data from this study with data obtained during phase I studies.
10.3.4.
Pharmacodynamic and Pharmacogenomic Analyses
The presence and the abundance of the translocation in blood will be correlated to the 
outcome of the patient, in search for a marker of response to the treatment or in 
anticipation of the progression. Also, the molecular parameters found in the tumor and 
blood samples will be correlated with the patients’ clinical results.
11.
ADMINISTRATIVE SECTION
11.1.
ETHICS
This clinical trial will be conducted in accordance with the ethical principles that have 
their origin in the World Medical Association (WMA) Declaration of Helsinki (see 
Appendix 5) and will be consistent with GCP guidelines and pertinent regulatory 
requirements.
The study personnel involved in conducting this trial will be qualified by education, 
training and experience to perform their respective task(s).
The study will be conducted in compliance with the protocol. The protocol, any 
amendments and the patient informed consent will receive IEC/IRB approval/favorable 
opinion prior to initiation, according to pertinent regulations.
The decision of the IEC/IRB concerning the conduct of the study will be made in 
writing to the Investigator, and a copy of this decision will be provided to the 
Sponsor(s) before the beginning of the study.
The Investigator and/or the Sponsor(s) is/are responsible for keeping the IEC/IRB 
informed of any significant new information about the study drug.
All protocol amendments will be agreed upon by the Sponsor(s) and the Investigator.
Administrative changes of the protocol are minor corrections and/or clarifications that 
have no impact on the way the study is to be conducted.


11.2.
MONITORING, AUDITING AND INSPECTING
The study will be monitored by regular site visits and telephone calls to the Investigator 
by the PharmaMar clinical trial monitor.
During site visits, the trial monitor should revise original patient records, drug 
accountability records and document retention (study file). Additionally, the trial 
monitor should observe study procedures and will discuss any problems with the 
Investigator.
Adequate time for these visits should be allocated by the Investigator. The Investigator 
should also ensure that the monitor is given direct access (as per International 
Conference on Harmonization [ICH] Topic E6 [R1] Guideline for Good Clinical 
Practice, Sections 4.9.7 and 6.10) to source documents (e.g., hospital or private charts, 
original laboratory records, appointment books, etc.) of the patient which support data 
entered in the case report forms, as defined in the ICH Topic E6 (R1) Guideline for 
Good Clinical Practice, Sections 1.51 and 1.52.
Systems and procedures will be implemented to ensure the quality of every aspect of the 
trial.
During the course of the trial, the Clinical Quality Assurance Department of the 
Sponsor(s) or external auditors contracted by the Sponsor(s) may conduct an onsite 
audit visit (ICH Topic E6 (R1) Guideline for Good Clinical Practice, Section 1.6).
Participation in this trial implies acceptance of potential inspection by national or 
foreign health authorities.
11.3.
PATIENT INFORMED CONSENT FORM
The rights, safety and well-being of the trial patients are the most important 
considerations and should prevail over interests of science and society.
The Informed Consent Forms will include all elements required by ICH, GCP and 
applicable regulatory requirements.
Prior to inclusion into the trial, the Investigator or a person designated by the 
Investigator must provide the patient and/or his/her representative with one copy of the 
Informed Consent Form for the clinical trial and one copy of the Informed Consent 
Form for the substudy. Both copies must provide written fully information about the 
clinical trial and the substudy in a language that is non-technical and easily understood. 
The Investigator should allow time necessary for the patient and/or his/her legally 
acceptable representative to inquire about the details of the clinical trial and the 
substudy. Each Informed Consent Form must be freely signed and personally dated by
the patient and/or his/her representative and by the person who conducted the informed 
consent discussion before the beginning of the study. The patient and/or representative 
should receive a copy of both the trial and the substudy signed Informed Consent
Forms, and any other written information provided to study patients and their 
representatives prior to participation in the trial.
During a patient's participation in the trial, any updates to the consent forms and any 
updates to the written information will be provided to him/her.
If there is a need to obtain new consent from the patients, the Investigator or a person 
designated by the Investigator should inform the patients of any new information 
relevant to the patients’ willingness to continue participation in the study, before 
obtaining the written consent.


11.4.
CONFIDENTIALITY / IDENTIFICATION OF PATIENTS
The collection and processing of personal data from the patients enrolled in this clinical 
trial will be limited to those data that are necessary to investigate the efficacy, safety, 
quality and usefulness of the study drug used in this trial.
It is the Investigator’s responsibility that sufficient information on the identity of the 
patients will be retained.
The trial monitor, the Sponsor(s)’s auditor, the IECs/IRBs and the regulatory authorities 
should have direct access to all requested trial-related records, and agree to keep the 
identity of study patients confidential.
The data must be collected and processed with adequate precautions to ensure 
confidentiality and compliance with applicable data privacy protection laws and 
regulations.
Explicit consent for the processing of personal data will be obtained from the 
participating patient before data collection, and this consent should also address the 
transfer of the data to other entities and countries.
The Sponsor(s) shall comply with the Directive 95/46/EEC of the European Parliament 
and of the Council of 24 October 1995 on the protection of individuals with regard to 
the processing of personal data and on the free movement of such data.
11.5.
CASE REPORT FORMS
Case report forms (CRFs) will be used to record all data for each patient. It is the 
responsibility of the Investigator to ensure that the CRFs are properly and completely 
filled in. CRFs must be completed for all patients who have given their informed 
consent (by themselves or through their representatives) and have been enrolled into the 
study.
A patient’s source documentation is the physician's patient records, and as such they 
should be maintained at the study site.
The data collected in the CRF will be entered into the Sponsor(s) databases, which 
comply with the Spanish Act implementing the Directive 95/46/EC of the European 
Parliament and of the Council of 24 October 1995 on the protection of individuals with 
regard to the processing of personal data.
11.6.
INSURANCE
The Sponsor(s) will provide insurance or indemnity in accordance with applicable 
regulatory requirements.
11.7.
RETENTION OF RECORDS
The Investigator/Institution should maintain trial documents according to Section 8 of 
the ICH Topic E6 (R1) Guideline for Good Clinical Practice and as required by 
applicable regulatory requirements.
Essential documents should be retained as per the aforementioned ICH guideline or for 
a longer period of time, if required by the applicable regulations. 


11.8.
USE OF INFORMATION AND PUBLICATIONS
Before the investigators of this study submit a paper or abstract for publication or 
otherwise publicly disclose information concerning the study drug or products, 
PharmaMar must be provided with at least 60 days to revise and approve the proposed 
publication or disclosure to ensure that confidential and proprietary data are protected.
If PharmaMar determines that patentable patient matter is disclosed in the proposed 
publication or disclosure, the publication or disclosure will be withheld for a period of 
time considered convenient. If the study is part of a multicenter study, the first 
publication of the study shall be made in conjunction with the presentation of a joint, 
multicenter publication of the study results with the investigators and the institutions 
from all appropriate sites that are contributing data, analysis and comments. However, if 
such a multicenter publication is not submitted within 12 months after conclusion, 
abandonment or termination of the study at all sites, the present study may be published 
individually in accordance with the procedure established above.
The order of the coauthors will reflect the relative contribution of each one to study 
development and analysis. In general, the first author will be the Investigator who 
recruits the highest number of patients with information finally available for data 
analysis. Relevant PharmaMar personnel who have fully participated in the study must 
be considered for co-authorship of the publication.


12.
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17. Guirouilh-Barbat J, Antony S, Pommier Y. Zalypsis (PM00104) is a potent inducer 
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27. Schoffski P, Dumez H, Wolter P, Stefan C, Wozniak A, Jimeno J, et al. Clinical 
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28. Grosso F, Sanfilippo R, Virdis E, Piovesan C, Collini P, Dileo P, et al. Trabectedin 
in myxoid liposarcomas (MLS): a long-term analysis of a single-institution series. Ann 
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29. Grosso F, Jones RL, Demetri GD, Judson IR, Blay JY, Le Cesne A, et al. Efficacy 
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Trabectedin (T) in metastatic Ewing's family tumors (EFT) patients (pts) progressing 
after standard chemotherapy. J Clin Oncol (Meeting Abstracts) 
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Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors. Mol 
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34. Griffin LB, Grohar P, Pommier Y, Khanna C, Khan J, Helman LJ. ET-743 
modulates EWS-FLi1 activity in Ewing’s sarcoma cells. In: Proceedings of the 100th 
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mirnor groove DNA binders. In; Manuscript in preparation.
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Use of epoetin and darbepoetin in patients with cancer: 2007 American Society of 
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diarrhea. J Clin Oncol 2004;22(14):2918-26.


Appendix 1. ECOG Performance Status Assessment Scale
Grade
ECOG PS*
0
Fully active, able to carry on all pre-disease performance without restriction
1
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work
2
Ambulatory and capable of all self-care but unable to carry out any work activities. 
Up and about more than 50% of waking hours
3
Capable of only limited self-care, confined to bed or chair more than 50% of 
waking hours
4
Completely disabled. Cannot carry on any self-care. Totally confined to bed or 
chair
5
Dead
*As published in Am. J. Clin. Oncol 5:649-655, 1982: Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., 
Davis, T.E., McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative 
Oncology Group. 


Appendix 2. Percent of Bone Marrow Irradiated
Percent of Normal Bone Marrow Irradiated Using Standard Radiation Ports
(From Ellis RE: Phys Med Biol 5:255, 1961)
Port
Marrow Volume at Risk 
(%)
Skull (not including mandible)
12
Upper limp girdle (unilateral, including the humeral head, 
scapulae, clavicle)
4
Sternum
2
Ribs (all)
8
Ribs (hemithorax)
4
Cervical vertebra (all)
3
Thoracic vertebra (all)
14
Lumbar vertebra (all)
11
Sacrum
14
Pelvis
26
Mantle
25
Upper para-aortic nodes
11
Inverted Y
45


Appendix 3. Response Evaluation Criteria in Solid Tumors (RECIST) v. 1.1
This document summarizes the main information contained in RECIST v. 1.1. 
Further details can be found in the original article: Eisenhauer EA, Therasse P, Bogaerts 
J, et al.: New response evaluation criteria in solid tumours: revised RECIST guideline (v. 
1.1). Eur J Cancer 2009; 45(2): 228-247.1
LIST OF ABBREVIATIONS
CR
Complete Response
CRF
Case Report Form
CT
Computerized Tomography
FDG-PET
Fluorodeoxyglucose-Positron Emission Tomography
MRI
Magnetic Resonance Imaging
NE
Not Evaluable
PD
Progressive Disease
PET
Positron Emission Tomography
PFS
Progression-free Survival
PR
Partial Response
PSA
Prostate-specific Antigen
RECIST
Response Evaluation Criteria in Solid Tumors
SD
Stable Disease
TTP
Time to Progression
LIST OF TABLES
Table 1. Time point response: patients with target (+/–non-target) disease.
Table 2. Time point response: patients with non-target disease only.
Table 3.  Best overall response when confirmation of complete response (CR) and partial 
response (PR) is  required.
Table 4. Summary of major changes from RECIST 1.0 to RECIST 1.12.
                                                
1 A summary of major changes from RECIST 1.0 to RECIST 1.1 can be found at the end of this document 
(Table 4).
2 This table is named Appendix I in the original RECIST 1.1 article.


1. MEASURABILITY OF TUMOR LESIONS AT BASELINE
1.1 Definitions
At baseline, tumor lesions/lymph nodes will be categorized measurable or non-measurable 
as follows:
1.1.1 Measurable
Tumor Lesions:
Must be accurately measured in at least one dimension (longest diameter in the plane of 
measurement is to be recorded) with a minimum size of: 

10 mm by computerized tomography (CT) scan (irrespective of scanner type) 
and magnetic resonance imaging (MRI) (no less than double the slice thickness 
and a minimum of 10 mm).

10 mm caliper measurement by clinical exam (when superficial). 

20 mm by chest X-ray (if clearly defined and surrounded by aerated lung).
Malignant Lymph Nodes:
To be considered pathologically enlarged and measurable, a lymph node must be ≥ 15 mm 
in short axis when assessed by CT scan (CT scan slice thickness recommended to be no 
greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and 
followed (see Schwartz et al. Eur J Cancer. 2009; 45(2):261-267). See also notes below on 
‘Baseline documentation of target and non-target lesions’ for information on lymph node 
measurement. 
1.1.2 Non-measurable
All other lesions, including small lesions (longest diameter < 10 mm or pathological lymph 
nodes with ≥ 10 to < 15 mm short axis) as well as lesions considered truly non-measurable. 
Lesions considered truly non-measurable include: leptomeningeal disease, ascites, pleural 
or pericardial effusion, inflammatory breast disease, lymphangitic involvement of skin or 
lung, and abdominal masses/abdominal organomegaly identified by physical exam that is 
not measurable by reproducible imaging techniques. 
1.1.3 Special Considerations Regarding Lesion Measurability
Bone lesions, cystic lesions, and lesions previously treated with local therapy require 
particular comment:
Bone Lesions:

Bone scan, positron emission tomography (PET) scan or plain films are not 
considered adequate imaging techniques to measure bone lesions. However, these 
techniques can be used to confirm the presence or disappearance of bone lesions. 

Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft tissue 
components, that can be evaluated by cross sectional imaging techniques such as CT 
or MRI can be considered as measurable lesions if the soft tissue component meets 
the definition of measurability described above.



Blastic bone lesions are non-measurable.
Cystic Lesions:

Lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since 
they are, by definition, simple cysts.

‘Cystic lesions’ thought to represent cystic metastases can be considered as 
measurable lesions, if they meet the definition of measurability described above. 
However, if non-cystic lesions are present in the same patient, these are preferred 
for selection as target lesions.
Lesions with Prior Local Treatment:

Tumor lesions situated in a previously irradiated area, or in an area subjected to 
other loco-regional therapy, are usually not considered measurable unless there has 
been demonstrated progression in the lesion. Study protocols should detail the 
conditions under which such lesions would be considered measurable.
1.2. Specifications by Methods of Measurement
1.2.1 Measurement of Lesions
All measurements should be recorded in metric notation, using calipers if clinically 
assessed. All baseline evaluations should be performed as close as possible to the treatment 
start and never more than four weeks before the beginning of the treatment.
1.2.2 Method of Assessment
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging based 
evaluation should always be done rather than clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam.
Clinical Lesions: 
Clinical lesions will only be considered measurable when they are superficial and ≥ 10 mm 
diameter as assessed using calipers (e.g., skin nodules). For the case of skin lesions, 
documentation by color photography including a ruler to estimate the size of the lesion is 
suggested. As noted above, when lesions can be evaluated by both clinical exam and 
imaging, imaging evaluation should be undertaken since it is more objective and may also 
be reviewed at the end of the study.
Chest X-Ray: 
Chest CT is preferred over chest X-ray, particularly when progression is an important 
endpoint, since CT is more sensitive than X-ray, particularly in identifying new lesions. 
However, lesions on chest X-ray may be considered measurable if they are clearly defined 
and surrounded by aerated lung. See original article, Appendix II, for more details.
Computed Tomography (CT), Magnetic Resonance Imaging (MRI):


CT is the best currently available and reproducible method to measure lesions selected for 
response assessment. This guideline has defined measurability of lesions on CT scan based 
on the assumption that CT slice thickness is 5 mm or less. As is described in original article 
(Appendix II), when CT scans have slice thickness greater than 5 mm, the minimum size 
for a measurable lesion should be twice the slice thickness. MRI is also acceptable in 
certain situations (e.g., for body scans). More details concerning the use of both CT and 
MRI for assessment of objective tumor response evaluation are provided in the original 
article, Appendix II.
Ultrasound:
Ultrasound is not useful in assessment of lesion size and should not be used as a method of 
measurement. Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to 
the next (described in greater detail in the original article, Appendix II). If new lesions are 
identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. 
If there is concern about radiation exposure at CT, MRI may be used instead of CT in 
selected instances.
Endoscopy, Laparoscopy: 
The use of these techniques for objective tumor evaluation is not advised. However, they 
can be useful to confirm complete pathological response when biopsies are obtained or to 
determine relapse in trials where recurrence following complete response or surgical 
resection is an endpoint.
Tumor Markers: 
Tumor markers alone cannot be used to assess objective tumor response. If markers are 
initially above the upper normal limit, however, they must normalize for a patient to be 
considered in complete response (CR). Because tumor markers are disease specific, 
instructions for their measurement should be incorporated into protocols on a disease 
specific basis. Specific guidelines for both CA-125 response (in recurrent ovarian cancer) 
and prostate-specific antigen (PSA) response (in recurrent prostate cancer) have been 
published (Rustin et al. J Natl Cancer Inst 2004; 96:487–488; Bubley et al. J Clin Oncol 
1999; 17:3461–3467; Scher et al. J Clin Oncol 2008; 26:1148–1159). In addition, the 
Gynecologic Cancer Intergroup has developed CA-125 progression criteria, which are to be 
integrated with objective tumor assessment for use in first-line trials in ovarian cancer 
(Vergote et al. J Natl Cancer Inst 2000; 92:1534–1535).
Cytology, Histology: 
These techniques can be used to differentiate between partial response (PR) and CR in rare 
cases if required by protocol (for example, residual lesions in tumor types such as germ cell 
tumors, where known residual benign tumors can remain). When effusions are known to be 
a potential adverse effect of treatment (e.g., with certain taxane compounds or angiogenesis 
inhibitors), the cytological confirmation of the neoplastic origin of any effusion that 
appears or worsens during treatment can be considered if the measurable tumor has met 


criteria for response or stable disease in order to differentiate between response (or stable 
disease) and progressive disease.
2. TUMOR RESPONSE EVALUATION
2.1 Assessment of Overall Tumor Burden and Measurable Disease
To assess objective response or future progression, it is necessary to estimate the overall 
tumor burden at baseline and use this as a comparator for subsequent measurements. Only 
patients with measurable disease at baseline should be included in protocols where 
objective tumor response is the primary endpoint. Measurable disease is defined by the 
presence of at least one measurable lesion (as detailed above in Section 1. Measurability of 
tumor at baseline). In studies where the primary endpoint is tumor progression (either time 
to progression or proportion with progression at a fixed date), the protocol must specify if 
entry is restricted to those with measurable disease or whether patients having non-
measurable disease only are also eligible.
2.2 Baseline Documentation of “Target” and “Non-target” Lesions
When more than one measurable lesion is present at baseline, all lesions up to a maximum 
of ve lesions total (and a maximum of two lesions per organ) representative of all 
involved organs should be identied as target lesions and will be recorded and measured at 
baseline (this means in instances where patients have only one or two organ sites involved 
that a maximum of two and four lesions will be recorded, respectively). For evidence to 
support the selection of only ve target lesions, see analyses on a large prospective 
database in the article by Bogaerts et al. Eur J Cancer 2009;45:248–260. 
Target lesions should be selected on the basis of their size (lesions with the longest 
diameter), be representative of all involved organs, but in addition should be those that lend 
themselves to reproducible repeated measurements. It may be the case that, on occasion, 
the largest lesion does not lend itself to reproducible measurement, in which circumstance 
the next largest lesion which can be measured reproducibly should be selected. To illustrate 
this point see the example in the original article, Figure 3 of Appendix II. 
Lymph nodes merit special mention since they are normal anatomical structures which may 
be visible by imaging even if not involved by tumor. As noted in the previous section, 
pathological nodes which are defined as measurable and may be identified as target lesions 
must meet the criterion of a short axis of ≥ 15 mm by CT scan. Only the short axis of these 
nodes will contribute to the baseline sum. The short axis of the node is the diameter 
normally used by radiologists to judge if a node is involved by solid tumor. Nodal size is 
normally reported as two dimensions in the plane in which the image is obtained (for CT 
scan this is almost always the axial plane; for MRI the plane of acquisition may be axial, 
saggital or coronal). The smaller of these measures is the short axis. For example, an 
abdominal node which is reported as being 20 mm x 30 mm has a short axis of 20 mm and 
qualifies as a malignant, measurable node. In this example, 20 mm should be recorded as 
the node measurement (see also the example in the original article, Figure 4 of Appendix 
II). All other pathological nodes (those with short axis ≥ 10 mm but < 15 mm) should be 


considered non-target lesions. Nodes that have a short axis < 10 mm are considered non-
pathological and should not be recorded or followed 
A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all 
target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes 
are to be included in the sum, then as noted above, only the short axis is added into the sum. 
The baseline sum diameters will be used as reference to further characterize any objective 
tumor regression in the measurable dimension of the disease.
All other lesions (or sites of disease) including pathological lymph nodes should be 
identified as non-target lesions and should also be recorded at baseline. Measurements are 
not required and these lesions should be followed as ‘present’, ‘absent’, or in rare cases 
‘unequivocal progression’ (more details to follow). In addition, it is possible to record 
multiple non-target lesions involving the same organ as a single item on the case record 
form (e.g., ‘multiple enlarged pelvic lymph nodes’ or ‘multiple liver metastases’).
2.3 Response Criteria
This section provides the definitions of the criteria used to determine objective tumor 
response for target lesions.
2.3.1 Evaluation of Target Lesions
Complete Response (CR):
Disappearance of all target lesions. Any pathological lymph 
nodes   (whether target or non-target) must have reduction in 
short axis to < 10 mm.
Partial Response (PR):
At least a 30% decrease in the sum of diameters of target 
lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD):
At least a 20% increase in the sum of diameters of target 
lesions, taking as reference the smallest sum on study (this 
includes the baseline sum if that is the smallest on study). In 
addition to the relative increase of 20%, the sum must also 
demonstrate an absolute increase of at least 5 mm. (Note: the 
appearance of one or more new lesions is also considered 
progression). 
Stable Disease (SD):
Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD taking as reference the smallest 
sum diameters while on study.


2.3.2 Special Notes on the Assessment of Target Lesions
Lymph Nodes:
Lymph nodes identified as target lesions should always have the actual short axis 
measurement recorded (measured in the same anatomical plane as the baseline 
examination), even if the nodes regress to below 10 mm on study. This means that when 
lymph nodes are included as target lesions, the ‘sum’ of lesions may not be zero even if 
complete response criteria are met, since a normal lymph node is defined as having a short 
axis of < 10 mm. Case report forms (CRFs) or other data collection methods may therefore 
be designed to have target nodal lesions recorded in a separate section where, in order to 
qualify for CR, each node must achieve a short axis < 10 mm. For PR, SD and PD, the 
actual short axis measurement of the nodes is to be included in the sum of target lesions.
Target Lesions that Become ‘Too Small to Measure’:
While on study, all lesions (nodal and non-nodal) recorded at baseline should have their 
actual measurements recorded at each subsequent evaluation, even when very small (e.g., 2 
mm). However, sometimes lesions or lymph nodes which are recorded as target lesions at 
baseline become so faint on CT scan that the radiologist may not feel comfortable assigning 
an exact measure and may report them as being ‘too small to measure’. When this occurs it 
is important that a value be recorded on the CRF. If it is the opinion of the radiologist that 
the lesion has likely disappeared, the measurement should be recorded as 0 mm. If the 
lesion is believed to be present and is faintly seen but too small to measure, a default value 
of 5 mm should be assigned (Note: It is less likely that this rule will be used for lymph 
nodes since they usually have a definable size when normal and are frequently surrounded
by fat such as in the retroperitoneum; however, if a lymph node is believed to be present 
and is faintly seen but too small to measure, a default value of 5 mm should be assigned in 
this circumstance as well). This default value is derived from the 5 mm CT slice thickness 
(but should not be changed with varying CT slice thickness). The measurement of these 
lesions is potentially non-reproducible, therefore providing this default value will prevent 
false responses or progressions based upon measurement error. To reiterate, however, if the 
radiologist is able to provide an actual measure, that should be recorded, even if it is below 
5 mm.
Lesions that Split or Coalesce on Treatment:
As noted in the original article, Appendix II, when non-nodal lesions ‘fragment’, the 
longest diameters of the fragmented portions should be added together to calculate the 
target lesion sum. Similarly, as lesions coalesce, a plane between them may be maintained 
that would aid in obtaining maximal diameter measurements of each individual lesion. If 
the lesions have truly coalesced such that they are no longer separable, the vector of the 
longest diameter in this instance should be the maximal longest diameter for the ‘coalesced 
lesion’.
2.3.3 Evaluation of Non-target Lesions
This section provides the definitions of the criteria used to determine the tumor response for 
the group of non-target lesions. While some non-target lesions may actually be measurable, 


they need not be measured and instead should be assessed only qualitatively at the time 
points specified in the protocol.
Complete Response (CR):
Disappearance of all non-target lesions and normalization of 
tumor marker level. All lymph nodes must be non-
pathological in size (< 10 mm short axis).
Non-CR/Non-PD:
Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits. 
Progressive Disease (PD):
Unequivocal progression (see comments below) of existing 
non-target lesions (Note: the appearance of one or more new 
lesions is also considered progression).
2.3.4 Special Notes on Assessment of Progression of Non-target Disease
The concept of progression of non-target disease requires additional explanation as follows:
When the Patient Also Has Measurable Disease:
In this setting, to achieve ‘unequivocal progression’ on the basis of the non-target disease, 
there must be an overall level of substantial worsening in non-target disease such that, even 
in presence of SD or PR in target disease, the overall tumor burden has increased 
sufficiently to merit discontinuation of therapy (see examples in the original article, 
Appendix II and further details below). A modest ‘increase’ in the size of one or more non-
target lesions is usually not sufficient to quality for unequivocal progression status. The 
designation of overall progression solely on the basis of change in non-target disease in the 
face of SD or PR of target disease will therefore be extremely rare.
When the Patient Has only Non-measurable Disease:
This circumstance arises in some phase III trials when it is not a criterion of study entry to 
have measurable disease. The same general concepts apply here as noted above, however, 
in this instance there is no measurable disease assessment to factor into the interpretation of 
an increase in non-measurable disease burden. Because worsening in non-target disease 
cannot be easily quantified (by definition: if all lesions are truly non-measurable) a useful 
test that can be applied when assessing patients for unequivocal progression is to consider if 
the increase in overall disease burden based on the change in non-measurable disease is 
comparable in magnitude to the increase that would be required to declare PD for 
measurable disease: i.e., an increase in tumor burden representing an additional 73% 
increase in ‘volume’ (which is equivalent to a 20% increase diameter in a measurable 
lesion). Examples include an increase in a pleural effusion from ‘trace’ to ‘large’, an 
increase in lymphangitic disease from localized to widespread, or may be described in 
protocols as ‘sufficient to require a change in therapy’. Some illustrative examples are 
shown in the original article, Figures 5 and 6 of Appendix II. If ‘unequivocal progression’ 
is seen, the patient should be considered to have had overall PD at that point. While it 
would be ideal to have objective criteria to apply to non-measurable disease, the very 
nature of that disease makes it impossible to do so, therefore the increase must be 
substantial.


2.3.5 New Lesions
The appearance of new malignant lesions denotes disease progression; therefore, some 
comments on detection of new lesions are important. There are no specic criteria for the 
identication of new radiographic lesions; however, the nding of a new lesion should be 
unequivocal: i.e., not attributable to differences in scanning technique, change in imaging 
modality or ndings thought to represent something other than tumor (for example, some 
‘new’ bone lesions may be simply healing or are of pre-existing lesions). This is 
particularly important when the patient’s baseline lesions show PR or CR. For example, 
necrosis of a liver lesion may be reported on a CT scan report as a ‘new’ cystic lesion, 
which it is not. 
A lesion identied on a follow-up study in an anatomical location that was not scanned at 
baseline is considered a new lesion and will indicate disease progression. An example of 
this is the patient who has visceral disease at baseline and while on study has a CT or MRI 
brain ordered which reveals metastases. The patient’s brain metastases are considered to be 
evidence of PD even if he/she did not have brain imaging at baseline. 
If a new lesion is equivocal, for example because of its small size, continued therapy and 
follow-up evaluation will clarify if it represents truly new disease. If repeat scans conrm 
there is denitely a new lesion, then progression should be declared using the date of the 
initial scan. 
While 
fluorodeoxyglucose-positron 
emission 
tomography 
(FDG-PET) 
response 
assessments need additional study, it is sometimes reasonable to incorporate the use of 
FDG-PET scanning to complement CT scanning in assessment of progression (particularly 
possible ‘new’ disease). New lesions on the basis of FDG-PET imaging can be identied 
according to the following algorithm: 
a.
Negative FDG-PET at baseline, with a positive3 FDG-PET at follow-up is a sign of 
PD based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up: 
o
If the positive FDG-PET at follow-up corresponds to a new site of disease 
conrmed by CT, this is PD.
o
If the positive FDG-PET at follow-up is not conrmed as a new site of 
disease on CT, additional follow-up CT scans are needed to determine if 
there is truly progression occurring at that site (if so, the date of PD will be 
the date of the initial abnormal FDG-PET scan). 
o
If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, 
this is not PD.
                                                
3 A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of 
the surrounding tissue on the attenuation corrected image.


2.4 Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the study treatment 
until the end of treatment taking into account any requirement for conrmation. On 
occasion a response may not be documented until after the end of therapy so protocols 
should be clear if post-treatment assessments are to be considered in determination of best 
overall response. Protocols must specify how any new therapy introduced before 
progression will affect best response designation. The patient’s best overall response 
assignment will depend on the ndings of both target and non-target disease and will also 
take into consideration the appearance of new lesions. Furthermore, depending on the 
nature of the study and the protocol requirements, it may also require conrmatory 
measurement (see Section 2.6. Confirmatory Measurement/Duration of Response). 
Specically, in non-randomized trials where response is the primary endpoint, conrmation 
of PR or CR is needed to deem either one the ‘best overall response’. This is described 
further below. 
2.4.1 Time Point Response 
It is assumed that at each protocol specied time point, a response assessment occurs. 
Table 1 provides a summary of the overall response status calculation at each time point 
for patients who have measurable disease at baseline. 
Table 1. Time point response: patients with target (+/–non-target) disease.
Target lesions
Non-target lesions
New
lesions
Overall
response
CR
CR
No
CR
CR
Non-CR/non-PD
No
PR
CR
Not evaluated
No
PR
PR
Non-PD or not all evaluated
No
PR
SD
Non-PD or not all evaluated
No
SD
Not all evaluated
Non- PD
No
NE
PD
Any
Yes or No
PD
Any
PD
Yes or No
PD
Any
Any
Yes
PD
CR, complete response; NE, inevaluable; PD, progressive disease;  PR, partial response; SD, 
stable disease. 
When patients have non-measurable (therefore non-target) disease only, Table 2 is to be 
used. 
Table 2. Time point response: patients with non-target disease only.
Non-target lesions
New lesions
Overall response
CR
No
CR
Non-CR/non-PD
No
Non-CR/non-PDa
Not all evaluated
No
NE
Unequivocal PD
Yes or No
PD
Any
Yes
PD
CR, complete response, NE, inevaluable; PD, progressive disease. 
a ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since 
SD is increasingly used as endpoint for assessment of efficacy in some trials; so, to 
assign this category when no lesions can be measured is not advised.


2.4.2 Missing Assessments and Inevaluable Designation
When no imaging/measurement is done at all at a particular time point, the patient is not 
evaluable (NE) at that time point. If only a subset of lesion measurements are made at an 
assessment, usually the case is also considered NE at that time point, unless a convincing 
argument can be made that the contribution of the individual missing lesion(s) would not 
change the assigned time point response. This would be most likely to happen in the case of 
PD. For example, if a patient had a baseline sum of 50 mm with three measured lesions and 
at follow-up only two lesions were assessed, but those gave a sum of 80 mm, the patient 
will have achieved PD status, regardless of the contribution of the missing lesion.
2.4.3 Best Overall Response: All Time Points 
The best overall response is determined once all the data for the patient is known.
Best Response Determination in Trials Where Conrmation of Complete or Partial 
Response IS NOT Required: 
Best response in these trials is dened as the best response across all time points (for 
example, a patient who has SD at rst assessment, PR at second assessment, and PD on last 
assessment has a best overall response of PR). When SD is believed to be best response, it 
must also meet the protocol specied minimum time from baseline. If the minimum time is 
not met when SD is otherwise the best time point response, the patient’s best response 
depends on the subsequent assessments. For example, a patient who has SD at rst 
assessment, PD at second and does not meet minimum duration for SD, will have a best 
response of PD. The same patient lost to follow-up after the rst SD assessment would be 
considered inevaluable. 
Best Response Determination in Trials Where Conrmation of Complete or Partial 
Response IS Required: 
Complete or partial responses may be claimed only if the criteria for each are met at a 
subsequent time point as specied in the protocol (generally four weeks later). In this 
circumstance, the best overall response can be interpreted as in Table 3. 
Table 3.  Best overall response when confirmation of complete response (CR) and partial response 
(PR) is  required.
Overall 
response.
First time point
Overall response.
Subsequent time point
BEST overall response
CR
CR
CR
CR
PR
SD, PD or PRa
CR
SD
SD provided minimum criteria for SD duration met, 
otherwise, PD
CR
PD
SD provided minimum criteria for SD duration met, 
otherwise, PD
CR
NE
SD provided minimum criteria for SD duration met, 
otherwise NE
PR
CR
PR
PR
PR
PR
PR
SD
SD
PR
PD
SD provided minimum criteria for SD duration met, 


Overall 
response.
First time point
Overall response.
Subsequent time point
BEST overall response
otherwise, PD
PR
NE
SD provided minimum criteria for SD duration met, 
otherwise NE
NE
NE
NE
CR, complete response; NE, inevaluable; PD, progressive disease; PR, partial response; SD, stable disease. 
a If a CR is truly met at first time point, then any disease seen at a subsequent time point, even disease meeting PR
criteria relative to baseline, makes the disease PD at that point (since disease must have reappeared after CR). Best 
response would depend on whether minimum duration for SD was met. However, sometimes ‘CR’ may be claimed 
when subsequent scans suggest small lesions were likely still present and in fact the patient had PR, not CR at the 
first time point. Under these circumstances, the original CR should be changed to PR and the best response is PR. 
2.4.4 Special Notes on Response Assessment
When nodal disease is included in the sum of target lesions and the nodes decrease to 
‘normal’ size (< 10 mm), they may still have a measurement reported on scans. This 
measurement should be recorded even though the nodes are normal in order not to overstate 
progression should it be based on increase in size of the nodes. As noted earlier, this means 
that patients with CR may not have a total sum of ‘zero’ on the CRF. 
In trials where conrmation of response is required, repeated ‘NE’ time point assessments 
may complicate best response determination. The analysis plan for the trial must address 
how missing data/assessments will be addressed in determination of response and 
progression. For example, in most trials it is reasonable to consider a patient with time 
point responses of PR-NE-PR as a conrmed response
Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be reported as 
‘symptomatic deterioration’. Every effort should be made to document objective 
progression even after discontinuation of treatment. Symptomatic deterioration is not a 
descriptor of an objective response: it is a reason for stopping study therapy. The objective 
response status of such patients is to be determined by evaluation of target and non-target 
disease as shown in Tables 1–3. 
Conditions that dene ‘early progression, early death and inevaluability’ are study specic 
and should be clearly described in each protocol (depending on treatment duration, 
treatment periodicity). 
In some circumstances it may be difcult to distinguish residual disease from normal tissue. 
When the evaluation of complete response depends upon this determination, it is 
recommended that the residual lesion be investigated (ne needle aspirate/biopsy) before 
assigning a status of complete response. FDG-PET may be used to upgrade a response to a 
CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is 
thought to represent brosis or scarring. The use of FDG-PET in this circumstance should 
be prospectively described in the protocol and supported by disease specic medical 
literature for the indication. However, it must be acknowledged that both approaches may 
lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity. 
For equivocal ndings of progression (e.g., very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions), treatment may continue until the next scheduled 


assessment. If at the next scheduled assessment, progression is conrmed, the date of 
progression should be the earlier date when progression was suspected. 
2.5 Frequency of Tumor Re-evaluation
Frequency of tumor re-evaluation while on treatment should be protocol specic and 
adapted to the type and schedule of treatment. However, in the context of phase II studies 
where the benecial effect of therapy is not known, follow-up every 6–8 weeks (timed to 
coincide with the end of a cycle) is reasonable. Smaller or greater time intervals than these 
could be justied in specic regimens or circumstances. The protocol should specify which 
organ sites are to be evaluated at baseline (usually those most likely to be involved with 
metastatic disease for the tumor type under study) and how often evaluations are repeated. 
Normally, all target and non-target sites are evaluated at each assessment. In selected 
circumstances certain non-target organs may be evaluated less frequently. For example, 
bone scans may need to be repeated only when CR is identied in target disease or when 
progression in bone is suspected. 
After the end of the treatment, the need for repetitive tumor evaluations depends on 
whether the trial has as a goal the response rate or the time to an event (progression/death). 
If ‘time to an event’ (e.g., time to progression, disease-free survival, progression-free 
survival) is the main endpoint of the study, then routine scheduled re-evaluation of protocol 
specied sites of disease is warranted. In randomized comparative trials in particular, the 
scheduled assessments should be performed as identied on a calendar schedule (for 
example: every 6–8 weeks on treatment or every 3–4 months after treatment) and should 
not be affected by delays in therapy, drug holidays or any other events that might lead to 
imbalance in a treatment arm in the timing of disease assessment. 
2.6 Conrmatory Measurement/Duration of Response
2.6.1 Conrmation
In non-randomized trials where response is the primary endpoint, conrmation of PR and 
CR is required to ensure responses identied are not the result of measurement error. This 
will also permit appropriate interpretation of results in the context of historical data where 
response has traditionally required conrmation in such trials (see Bogaerts et al. Eur J 
Cancer 2009; 45(2): 248-260). However, in all other circumstances, i.e., in randomized 
trials (phase II or III) or studies where stable disease or progression are the primary 
endpoints, conrmation of response is not required since it will not add value to the 
interpretation of trial results. However, elimination of the requirement for response 
conrmation may increase the importance of central review to protect against bias, in 
particular in studies which are not blinded. 
In the case of SD, measurements must have met the SD criteria at least once after study 
entry at a minimum interval (in general not less than 6–8 weeks) that is dened in the study 
protocol.


2.6.2 Duration of Overall Response
The duration of overall response is measured from the time measurement criteria are rst 
met for CR/PR (whichever is rst recorded) until the rst date that recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the smallest 
measurements recorded on study). 
The duration of overall complete response is measured from the time measurement criteria 
are rst met for CR until the rst date that recurrent disease is objectively documented. 
2.6.3 Duration of Stable Disease
Stable disease is measured from the start of the treatment (in randomized trials, from date 
of randomization) until the criteria for progression are met, taking as reference the smallest 
sum on study (if the baseline sum is the smallest, this is the reference for calculation of PD). 
The clinical relevance of the duration of SD varies in different studies and diseases. If the 
proportion of patients achieving SD for a minimum period of time is an endpoint of 
importance in a particular trial, the protocol should specify the minimal time interval 
required between two measurements for determination of stable disease. 
Note: The duration of response and SD as well as the progression-free survival are 
inuenced by the frequency of follow-up after baseline evaluation. It is not in the scope of 
this guideline to dene a standard follow-up frequency. The frequency should take into 
account many parameters including disease types and stages, treatment periodicity and 
standard practice. However, these limitations of the precision of the measured endpoint 
should be taken into account if comparisons between trials are to be made. 
2.7 Progression-free Survival/Proportion Progression-free
2.7.1 Phase II Trials
This guideline is focused primarily on the use of objective response endpoints for phase II 
trials. In some circumstances, ‘response rate’ may not be the optimal method to assess the 
potential anticancer activity of new agents/regimens. In such cases ‘progression-free 
survival’ (PFS) or the ‘proportion progression-free’ at landmark time points, might be 
considered appropriate alternatives to provide an initial signal of biologic effect of new 
agents. It is clear, however, that in an uncontrolled trial, these measures are subject to 
criticism since an apparently promising observation may be related to biological factors 
such as patient selection and not the impact of the intervention. Thus, phase II screening 
trials utilizing these endpoints are best designed with a randomized control. Exceptions 
may exist where the behavior patterns of certain cancers are so consistent (and usually 
consistently poor), that a non-randomized trial is justiable (see Van Glabbeke et al. Eur J 
Cancer 2002; 38(4): 543-549). However, in these cases it will be essential to document with 
care the basis for estimating the expected PFS or proportion progression-free in the absence 
of a treatment effect.


2.7.2 Phase III trials
Phase III trials in advanced cancers are increasingly designed to evaluate PFS or time to 
progression (TTP) as the primary outcome of interest. Assessment of progression is 
relatively straightforward if the protocol requires all patients to have measurable disease. 
However, restricting entry to this subset of patients is subject to criticism: it may result in a 
trial where the results are less likely to be generalizable if, in the disease under study, a 
substantial proportion of patients would be excluded. Moreover, the restriction to entry will 
slow recruitment to the study. Increasingly, therefore, trials allow entry of both patients 
with measurable disease as well as those with non-measurable disease only. In this 
circumstance, care must be taken to explicitly describe the ndings which would qualify for 
progressive disease for those patients without measurable lesions. Furthermore, in this 
setting, protocols must indicate if the maximum number of recorded target lesions for those 
patients with measurable disease may be relaxed from ve to three (based on the data found 
in Bogaerts et al. Eur J Cancer 2009; 45:248–260, and Moskowitz et al. Eur J Cancer 2009; 
45:300–310). As found in the ‘special notes on assessment of progression’ (Section 2.3.4), 
these guidelines offer recommendations for assessment of progression in this setting. 
Furthermore, if available, validated tumor marker measures of progression (as has been 
proposed for ovarian cancer) may be useful to integrate into the denition of progression. 
Centralized blinded review of imaging studies or of source imaging reports to verify 
‘unequivocal progression’ may be needed if important drug development or drug approval 
decisions are to be based on the study outcome. Finally, as noted earlier, because the date 
of progression is subject to ascertainment bias, timing of investigations in study arms 
should be the same. The article by Dancey et al. Eur J Cancer 2009; 45:281–289, provides 
a more detailed discussion of the assessment of progression in randomized trials.
2.8 Independent Review of Response and Progression
For trials where objective response (CR + PR) is the primary endpoint, and in particular 
where key drug development decisions are based on the observation of a minimum number 
of responders, it is recommended that all claimed responses be reviewed by an expert(s) 
independent of the study. If the study is a randomized trial, ideally reviewers should be 
blinded to treatment assignment. Simultaneous review of the patients’ les and radiological 
images is the best approach.
Independent review of progression presents some more complex issues: for example, there 
are statistical problems with the use of central-review-based progression time in place of 
investigator-based progression time due to the potential introduction of informative 
censoring when the former precedes the latter. An overview of these factors and other 
lessons learned from independent review is provided in an article by Ford et al. Eur J 
Cancer 2009; 45:268–274.


2.9 Reporting Best Response Results
2.9.1 Phase II Trials
When response is the primary endpoint, and thus all patients must have measurable disease 
to enter the trial, all patients included in the study must be accounted for in the report of the 
results, even if there are major protocol treatment deviations or if they are not evaluable. 
Each patient will be assigned one of the following categories: 
1. Complete response.
2. Partial response. 
3. Stable disease.
4. Progression.
5. Inevaluable for response: specify reasons [for example: early death, malignant 
disease; early death, toxicity; tumor assessments not repeated/incomplete; other 
(specify)]. 
Normally, all eligible patients should be included in the denominator for the calculation of 
the response rate for phase II trials (in some protocols it will be appropriate to include all 
treated patients). It is generally preferred that 95% two-sided condence limits are given for 
the calculated response rate. Trial conclusions should be based on the response rate for all 
eligible (or all treated) patients and should not be based on a selected ‘evaluable’ subset.
2.9.2 Phase III Trials
Response evaluation in phase III trials may be an indicator of the relative antitumor activity 
of the treatments evaluated and is almost always a secondary endpoint. Observed 
differences in response rate may not predict the clinically relevant therapeutic benet for 
the population studied. If objective response is selected as a primary endpoint for a phase 
III study (only in circumstances where a direct relationship between objective tumor 
response and a clinically relevant therapeutic benet can be unambiguously demonstrated 
for the population studied), the same criteria as those applying to phase II trials should be 
used and all patients entered should have at least one measurable lesion. 
In those many cases where response is a secondary endpoint and not all trial patients have 
measurable disease, the method for reporting overall best response rates must be pre-
specied in the protocol. In practice, response rate may be reported using either an ‘intent 
to treat’ analysis (all randomized patients in the denominator) or an analysis where only the 
subset of patients with measurable disease at baseline are included. The protocol should 
clearly specify how response results will be reported, including any subset analyses that are 
planned.
The original version of RECIST suggested that in phase III trials one could write protocols 
using a ‘relaxed’ interpretation of the RECIST guidelines (for example, reducing the 
number of lesions measured) but this should no longer be done since these revised 
guidelines have been amended in such a way that it is clear how these criteria should be 
applied for all trials in which anatomical assessment of tumor response or progression are 
endpoints. 


BIBLIOGRAPHIC REFERENCES4
Baar J, Tannock I. Analyzing the same data in two ways: a demonstration model to illustrate the 
reporting and misreporting of clinical trials. J Clin Oncol 1989;7:969–978.
Barrett T, Choyke PL, Kobayashi H. Imaging of the lymphatic system: new horizons. Contrast 
Media Mol Imaging 2006;1(6):230–245.
Bogaerts J, Ford R, Sargent D, et al. Individual patient data analysis to assess modifications to the 
RECIST criteria. Eur J Cancer 2009;45:248–260.
Bubley GJ, Carducci M, DahutW, et al. Eligibility and response guidelines for phase II clinical 
trials in androgen-independent prostate cancer: recommendations from the Prostate-Specific 
Antigen Working Group. J Clin Oncol 1999;17:3461–3467.
Buyse M, Thirion P, Carlson RW, et al. Relation between tumor response to first-line chemotherapy 
and survival in advanced colorectal cancer: a meta-analysis. Meta-analysis group in Cancer. Lancet 
2000;356:373–378.
Catalano C, Francone M, Ascarelli A, Mangia M, Iacucci I, Passariello R. Optimizing radiation 
dose and image quality. Eur Radiol 2007;17(Suppl 6):F26–F32. 
Cheson BD, Pfistner B, Juweid ME, et al. Revised response criteria for malignant lymphoma. J Clin 
Oncol 2007;10:579–586.
Dancey JE, Dodd LE, Ford R, et al. Recommendations for the assessment of progression in 
randomised cancer treatment trials. Eur J Cancer 2009;45:281–289. 
El-Maraghi RH, Eisenhauer EA. Review of phase II trial designs used in studies of molecular 
targeted agents: outcomes and predictors of success in phase III. J Clin Oncol 2008;10:1346–1354. 
Ford R, Schwartz L, Dancey J, et al. Lessons learned from independent central review. Eur J 
Cancer 2009;45:
268–274.
Goffin J, Baral S, Tu D, et al. Objective responses in patients with malignant melanoma or renal 
cell cancer in early clinical studies do not predict regulatory approval. Clin Cancer Res
2005;15:5928–5934.
Low RN. Abdominal MRI advances in the detection of liver tumours and characterization. Lancet 
Oncol 2007;8(6):525–535.
Macdonald DR, Cascino TL, Schold Jr SC, Cairncross JG. Response criteria for phase II studies of
supratentorial malignant glioma. J Clin Oncol 1990;8:1277–280.
                                                
4 All references contained in the original RECIST v.1.1 article are shown, although only some of them are 
mentioned in the current summarized document.


Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer
1981;47:207–214.
Moskowitz CS, Jia X, Schwartz LH, Go¨nen M. A simulation study to evaluate the impact of the 
number of lesions measured on response assessment. Eur J Cancer 2009;45:300–310.
Paesmans M, Sculier JP, Libert P, et al. Response to chemotherapy has predictive value for further 
survival of patients with advanced non-small cell lung cancer: 10 years experience of the European 
Lung Cancer Working Party. Eur J Cancer 1997;33:2326–2332.
Rustin GJ, Quinn M, Thigpen T, et al. Re: New guidelines to evaluate the response to treatment in 
solid tumors (ovarian cancer). J Natl Cancer Inst 2004;96:487–488.
Sargent D, Rubinstein L, Schwartz L, et al. Validation of novel imaging methodologies for use as 
cancer clinical trials end-points. Eur J Cancer 2009;45:290–299.
Scher H, Halabi S, Tannock I, et al. Design and end points of clinical trials for patients with 
progressive prostate cancer and castrate levels of testosterone: recommendations of the Prostate 
Cancer Clinical Trials Working Group. J Clin Oncol 2008;26:1148–1159.
Schwartz LH, Bogaerts J, Ford R, et al. Evaluation of lymph nodes with RECIST 1.1. Eur J Cancer
2009;45:261–267.
Shankar LK, Hoffman JM, Bacharach S, et al. National Cancer Institute. Consensus 
recommendations for the use of 18F-FDG PET as an indicator of therapeutic response in patients in 
National Cancer Institute Trials. J Nucl Med 2006;47(6):1059–1066.
Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment 
in solid tumors (RECIST Guidelines). J Natl Cancer Inst 2000;92:205–216.
Therasse P, Eisenhauer EA, Verweij J. RECIST revisited: a review of validation studies on tumour 
assessment. Eur J Cancer 2006;42:1031–1039.
Tonkin K, Tritchler D, Tannock I. Criteria of tumor response used in clinical trials of 
chemotherapy. J Clin Oncol 1985;3:870–875.
Van Glabbeke M, Verweij J, Judson I, Nielsen OS. EORTC Soft Tissue and Bone Sarcoma Group: 
Progression-free rate as the principal end-point for phase II trials in soft-tissue sarcomas. Eur J 
Cancer 2002;38:543–549.
Vergote I, Rustin GJ, Eisenhauer EA, et al. Re: new guidelines to evaluate the response to treatment 
in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup. J Natl Cancer Inst 2000;92:1534–
1535.


Table 4. Summary of major changes from RECIST 1.0 to RECIST 1.1.
RECIST v. 1.0
RECIST v. 1.1
Rationale
CT: 10 mm spiral 
20 mm non-spiral 
CT 10 mm; delete 
reference to 
spiral scan 
Most scans used have 5 mm or less slice 
thickness. Clearer to give instruction based 
on slice interval if it is greater than 5 mm 
Clinical: 20 mm 
Clinical: 10 mm (must 
be measurable with 
calipers) 
Caliper measurement will make this reliable 
Minimum size 
measurable 
lesions 
Lymph node: not 
mentioned 
CT: 
≥ 15 mm short axis for 
target 
≥ 10–<15 mm for non-
target 
< 10 mm is non-
pathological 
Since nodes are normal structure need to 
define pathological enlargement. Short axis 
is most sensitive 
Special considerations 
on 
lesion measurability 
–
Notes included on bone 
lesions, cystic lesions 
Clarify frequently asked questions 
Overall tumor burden 
10 lesions (5 per organ) 
5 lesions (2 per organ) 
Data warehouse analysis shows no loss of 
information if lesion number reduced from 
10 to 5. A maximum of 2 lesions per organ 
yields sufficient representation per disease 
site 
CR lymph node not 
mentioned 
CR lymph nodes must 
be <10 mm short axis 
In keeping with normal size of nodes 
Response criteria target 
disease 
PD 20% increase over 
smallest sum on 
study or new lesions 
PD 20% increase over 
smallest sum on study 
(including baseline if 
that is smallest) and 
at least 5 mm increase or 
new lesions 
Clarification that if baseline measurement is 
smaller than any on study measurement, it 
is reference against which PD is assessed 5 
mm absolute increase to guard against over  
calling PD when total sum is very small and 
20% increase is within measurement error 
Response criteria non-
target 
disease 
‘Unequivocal progression’ 
considered as PD 
More detailed 
description of 
‘unequivocal 
progression’ to 
indicate that it should 
not normally trump 
target disease 
status. It must be 
representative of overall 
disease status change, 
not a single lesion 
increase 
Confusion with RECIST 1.0 where some 
were considering PD if ‘increase’ in any 
non-target lesion, even when target disease 
is stable or responding 
New lesions 
–
New section on New 
lesions 
To provide guidance on when a lesion is 
considered new (and thus PD) 
Overall response 
Table integrated target and 
non-target 
lesions 
Two tables: one 
integrating target and 
non-target and the 
other of non-target only 
Special notes: 
How to assess and 
measure lymph nodes 
CR in face of residual 
tissue 
Discussion of 
‘equivocal’ 
progression 
To account for the fact that RECIST criteria 
are now being used in trials where PFS is 
the endpoint and not all patients have 
measurable (target) disease at baseline 
Frequently asked questions on these topics 


RECIST v. 1.0
RECIST v. 1.1
Rationale
Confirmatory measure For CR and PR: criteria 
must be met again 4 
weeks after initial 
documentation 
Retain this requirement 
ONLY for non-
randomized trials with  
primary endpoint of 
response 
Data warehouse shows that response rates 
rise when confirmation is eliminated, but 
the only circumstance where this is 
important is in trials where there is no 
concurrent comparative control and where 
this measure is the primary endpoint 
Progression-free 
survival 
General comments only 
More specific comments 
on 
use of PFS (or 
proportion 
progression-free) as 
phase II endpoint 
Greater detail on PFS 
assessment in phase III 
trials 
Increasing use of PFS in phase III trials 
requires guidance on assessment of PD in 
patients with non-measurable disease 
Reporting of response 
results 
9 categories suggested for 
reporting phase II results 
Divided into phase II 
and phase III 
9 categories collapsed 
into 5 
In phase III, guidance 
given about reporting 
response 
Simplifies reporting and clarifies how to 
report phase II and III data consistently 
Response in phase III 
trials 
More relaxed guidelines 
possible if protocol 
specified 
This section removed 
and referenced in 
section 
above: no need to have 
different criteria for
phase II and III 
Simplification of response assessment by 
reducing number of lesions and eliminating 
need for confirmation in randomized studies 
where response is not the primary endpoint 
makes separate ‘rules’ unnecessary 
Imaging appendix 
Appendix I 
Appendix II: updated 
with detailed guidance 
on use of MRI, PET/CT 
Other practical guidance 
included
Evolving use of newer modalities 
addressed. Enhanced guidance in response 
to frequent questions and from radiology 
review experience 
New appendices 
Appendix I: comparison 
of 
RECIST 1.0 and 1.1 
Appendix III: frequently 
asked questions
CR, complete response; CT, computed tomography; MRI, magnetic resonance imaging; RECIST, response evaluation 
criteria in solid tumors; PD, progressive disease; PET, positron emission tomography; PFS, progression-free survival; PR, 
partial response.


Appendix 4. Cockcroft and Gault’s formula
For calculating creatinine clearance* 
[(140-age (years)) x weight (Kg)]
Creatinine clearance (ml/min) =  
x G1
72 x serum creatinine (mg/dl)
[(140-age (years)) x weight (Kg)]
Creatinine clearance (ml/min) =  
xG1
72 x serum creatinine (mol/l) x 0.0113
1G (Gender)= 0.85 if Female; 1 if Male.
*As published in Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. 
Nephron. 1976;16(1):31-41.


Appendix 5. Declaration of Helsinki
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI
Ethical Principles for Medical Research Involving Human Subjects
Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, and amended 
by the:
29th WMA General Assembly, Tokyo, Japan, October 1975
35th WMA General Assembly, Venice, Italy, October 1983
41st WMA General Assembly, Hong Kong, September 1989
48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996
52nd WMA General Assembly, Edinburgh, Scotland, October 2000 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 
added)
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on paragraph 30 added)
59th WMA General Assembly, Seoul, October 2008
A. INTRODUCTION 
1. The World Medical Association (WMA) has developed the Declaration of Helsinki 
as a statement of ethical principles for medical research involving human subjects, 
including research on identifiable human material and data. 
The Declaration is intended to be read as a whole and each of its constituent 
paragraphs should not be applied without consideration of all other relevant 
paragraphs.
2. Although the Declaration is addressed primarily to physicians, the WMA 
encourages other participants in medical research involving human subjects to adopt 
these principles.
3. It is the duty of the physician to promote and safeguard the health of patients, 
including those who are involved in medical research. The physician's knowledge 
and conscience are dedicated to the fulfilment of this duty.
4. The Declaration of Geneva of the WMA binds the physician with the words, "The 
health of my patient will be my first consideration," and the International Code of 
Medical Ethics declares that, "A physician shall act in the patient's best interest 
when providing medical care."
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should 
be provided appropriate access to participation in research.
6. In medical research involving human subjects, the well-being of the individual 
research subject must take precedence over all other interests.
7. The primary purpose of medical research involving human subjects is to understand 


the causes, development and effects of diseases and improve preventive, diagnostic 
and therapeutic interventions (methods, procedures and treatments). Even the best 
current interventions must be evaluated continually through research for their 
safety, effectiveness, efficiency, accessibility and quality.
8. In medical practice and in medical research, most interventions involve risks and 
burdens.
9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are 
particularly vulnerable and need special protection. These include those who cannot 
give or refuse consent for themselves and those who may be vulnerable to coercion 
or undue influence.
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or 
regulatory requirement should reduce or eliminate any of the protections for 
research subjects set forth in this Declaration.
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH 
11. It is the duty of physicians who participate in medical research to protect the life, 
health, dignity, integrity, right to self-determination, privacy, and confidentiality of 
personal information of research subjects.
12. Medical research involving human subjects must conform to generally accepted 
scientific principles, be based on a thorough knowledge of the scientific literature, 
other relevant sources of information, and adequate laboratory and, as appropriate, 
animal experimentation. The welfare of animals used for research must be 
respected. 
13. Appropriate caution must be exercised in the conduct of medical research that may 
harm the environment.
14. The design and performance of each research study involving human subjects must 
be clearly described in a research protocol. The protocol should contain a statement 
of the ethical considerations involved and should indicate how the principles in this 
Declaration have been addressed. The protocol should include information 
regarding funding, sponsors, institutional affiliations, other potential conflicts of 
interest, incentives for subjects and provisions for treating and/or compensating 
subjects who are harmed as a consequence of participation in the research study. 
The protocol should describe arrangements for post-study access by study subjects 
to interventions identified as beneficial in the study or access to other appropriate 
care or benefits.
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee 
must be independent of the researcher, the Sponsor(s) and any other undue 
influence. It must take into consideration the laws and regulations of the country or 
countries in which the research is to be performed as well as applicable international 


norms and standards but these must not be allowed to reduce or eliminate any of the 
protections for research subjects set forth in this Declaration. The committee must 
have the right to monitor ongoing studies. The researcher must provide monitoring 
information to the committee, especially information about any serious adverse 
events. No change to the protocol may be made without consideration and approval 
by the committee.
16. Medical research involving human subjects must be conducted only by individuals 
with the appropriate scientific training and qualifications. Research on patients or 
healthy volunteers requires the supervision of a competent and appropriately 
qualified physician or other health care professional. The responsibility for the 
protection of research subjects must always rest with the physician or other health 
care professional and never the research subjects, even though they have given 
consent.
17. Medical research involving a disadvantaged or vulnerable population or community 
is only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population 
or community stands to benefit from the results of the research.
18. Every medical research study involving human subjects must be preceded by 
careful assessment of predictable risks and burdens to the individuals and 
communities involved in the research in comparison with foreseeable benefits to 
them and to other individuals or communities affected by the condition under 
investigation.
19. Every clinical trial must be registered in a publicly accessible database before 
recruitment of the first subject.
20. Physicians may not participate in a research study involving human subjects unless 
they are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of 
positive and beneficial results.
21. Medical research involving human subjects may only be conducted if the 
importance of the objective outweighs the inherent risks and burdens to the research 
subjects.
22. Participation by competent individuals as subjects in medical research must be 
voluntary. Although it may be appropriate to consult family members or community 
leaders, no competent individual may be enrolled in a research study unless he or 
she freely agrees.
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study 
on their physical, mental and social integrity.
24. In medical research involving competent human subjects, each potential subject 
must be adequately informed of the aims, methods, sources of funding, any possible 
conflicts of interest, institutional affiliations of the researcher, the anticipated 
benefits and potential risks of the study and the discomfort it may entail, and any 
other relevant aspects of the study. The potential subject must be informed of the 


right to refuse to participate in the study or to withdraw consent to participate at any 
time without reprisal. Special attention should be given to the specific information 
needs of individual potential subjects as well as to the methods used to deliver the 
information. After ensuring that the potential subject has understood the 
information, the physician or another appropriately qualified individual must then 
seek the potential subject's freely-given informed consent, preferably in writing. If 
the consent cannot be expressed in writing, the non-written consent must be 
formally documented and witnessed.
25. For medical research using identifiable human material or data, physicians must 
normally seek consent for the collection, analysis, storage and/or reuse. There may 
be situations where consent would be impossible or impractical to obtain for such 
research or would pose a threat to the validity of the research. In such situations the 
research may be done only after consideration and approval of a research ethics 
committee.
26. When seeking informed consent for participation in a research study the physician 
should be particularly cautious if the potential subject is in a dependent relationship 
with the physician or may consent under duress. In such situations the informed 
consent should be sought by an appropriately qualified individual who is completely 
independent of this relationship.
27. For a potential research subject who is incompetent, the physician must seek 
informed consent from the legally authorized representative. These individuals must 
not be included in a research study that has no likelihood of benefit for them unless 
it is intended to promote the health of the population represented by the potential 
subject, the research cannot instead be performed with competent persons, and the 
research entails only minimal risk and minimal burden.
28. When a potential research subject who is deemed incompetent is able to give assent 
to decisions about participation in research, the physician must seek that assent in 
addition to the consent of the legally authorized representative. The potential 
subject's dissent should be respected.
29. Research involving subjects who are physically or mentally incapable of giving 
consent, for example, unconscious patients, may be done only if the physical or 
mental condition that prevents giving informed consent is a necessary characteristic 
of the research population. In such circumstances the physician should seek 
informed consent from the legally authorized representative. If no such 
representative is available and if the research cannot be delayed, the study may 
proceed without informed consent provided that the specific reasons for involving 
subjects with a condition that renders them unable to give informed consent have 
been stated in the research protocol and the study has been approved by a research 
ethics committee. Consent to remain in the research should be obtained as soon as 
possible from the subject or a legally authorized representative.
30. Authors, editors and publishers all have ethical obligations with regard to the 
publication of the results of research. Authors have a duty to make publicly 
available the results of their research on human subjects and are accountable for the 
completeness and accuracy of their reports. They should adhere to accepted 


guidelines for ethical reporting. Negative and inconclusive as well as positive 
results should be published or otherwise made publicly available. Sources of 
funding, institutional affiliations and conflicts of interest should be declared in the 
publication. Reports of research not in accordance with the principles of this 
Declaration should not be accepted for publication.
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE
31. The physician may combine medical research with medical care only to the extent 
that the research is justified by its potential preventive, diagnostic or therapeutic 
value and if the physician has good reason to believe that participation in the 
research study will not adversely affect the health of the patients who serve as 
research subjects.
32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:
a. The use of placebo, or no treatment, is acceptable in studies where no current 
proven intervention exists; or 
b. Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
patients who receive placebo or no treatment will not be subject to any risk of 
serious or irreversible harm. Extreme care must be taken to avoid abuse of this 
option.
33. At the conclusion of the study, patients entered into the study are entitled to be 
informed about the outcome of the study and to share any benefits that result from 
it, for example, access to interventions identified as beneficial in the study or to 
other appropriate care or benefits.
34. The physician must fully inform the patient which aspects of the care are related to 
the research. The refusal of a patient to participate in a study or the patient's 
decision to withdraw from the study must never interfere with the patient-physician 
relationship.
35. In the treatment of a patient, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from 
the patient or a legally authorized representative, may use an unproven intervention 
if in the physician's judgment it offers hope of saving life, re-establishing health or 
alleviating suffering. Where possible, this intervention should be made the object of 
research, designed to evaluate its safety and efficacy. In all cases, new information
should be recorded and, where appropriate, made publicly available.

SAP_001.pdf: 
PM104-B-003-10 
Phase II Multicenter, Open-label, Clinical and Pharmacokinetic Study of 
Zalypsis® (PM00104) in Patients with Unresectable Locally Advanced 
and/or Metastatic Ewing Family of Tumors (EFT) Progressing After 
at Least One Prior Line of Chemotherapy 
Statistical Analysis Plan 
Version 
2.0 
Date 
07-02-2013 
NCT Code 01222767


 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS .................................................................................................................... 4 
1. 
INTRODUCTION ............................................................................................................................ 5 
1.1. 
STUDY RATIONALE ..................................................................................................................... 5 
1.2. 
INFORMATION ON STUDY DRUG .................................................................................................. 5 
2. 
OBJECTIVES ................................................................................................................................... 5 
2.1. 
PRIMARY .................................................................................................................................... 5 
2.2. 
SECONDARY ............................................................................................................................... 5 
3. 
STUDY DESIGN ............................................................................................................................... 6 
4. 
STUDY POPULATIONS ................................................................................................................. 6 
4.1. 
ANALYSIS POPULATIONS ............................................................................................................ 6 
4.1.1. 
Efficacy population ............................................................................................................... 6 
4.1.2. 
Safety population .................................................................................................................. 6 
5. 
ENDPOINTS ..................................................................................................................................... 6 
5.1. 
PRIMARY ENDPOINTS ................................................................................................................. 6 
5.2. 
SECONDARY ENDPOINTS ............................................................................................................ 6 
5.3. 
OTHER ENDPOINTS ..................................................................................................................... 7 
6. 
SAMPLE SIZE .................................................................................................................................. 8 
6.1. 
SAMPLE SIZE DETERMINATION ................................................................................................... 8 
7. 
STATISTICAL ANALYSIS ............................................................................................................ 8 
7.1. 
TREATMENT DISCONTINUATION AND PROTOCOL DEVIATIONS MANAGEMENT ............................ 8 
7.2. 
DEMOGRAPHIC ANALYSIS .......................................................................................................... 9 
7.3. 
STATISTICAL METHODOLOGY FOR EFFICACY .............................................................................. 9 
7.4. 
STATISTICAL METHODOLOGY FOR SAFETY ............................................................................... 10 
7.4.1. 
Exposure ............................................................................................................................. 10 
7.4.2. 
Cycle delays, skipped doses and dose modifications .......................................................... 10 
7.4.3. 
Adverse events .................................................................................................................... 11 
7.4.4. 
Deaths and serious adverse events ..................................................................................... 11 
7.4.5. 
Laboratory evaluations ....................................................................................................... 11 
7.4.6. 
Other safety evaluations ..................................................................................................... 12 
7.4.7. 
Concomitant Medication .................................................................................................... 12 
7.5. 
PHARMACOKINETIC (PK), PHARMACODYNAMIC PROFILE AND PHARMACOGENOMIC (PGX) 
EVALUATION .......................................................................................................................................... 12 
8. 
OTHER STATISTICAL ANALYSIS CONSIDERATIONS ...................................................... 12 
8.1. 
MISSING VALUE MANAGEMENT ................................................................................................ 12 
8.1.1. 
Imputation in incomplete dates ........................................................................................... 12 
8.2. 
DATA ANALYSIS CONVENTIONS ............................................................................................... 13 
8.3. 
MULTIVARIATE ANALYSES ....................................................................................................... 13 
8.4. 
FIXED, RANDOM OR MIXED EFFECTS MODELS ........................................................................... 13 
8.5. 
SUBGROUP ANALYSIS .............................................................................................................. 13 
8.6. 
INTERIM ANALYSIS .................................................................................................................. 13 
8.7. 
INDEPENDENT REVIEW COMMITTEE .......................................................................................... 13 
9. 
STATISTICAL SOFTWARE ........................................................................................................ 13 
10. 
APPENDIX I. PATIENT DISPOSITION AND PROTOCOL DEVIATIONS .................... 17 
10.1. 
GENERAL CHARACTERISTICS .................................................................................................... 17 
10.1.1. 
Patients disposition ........................................................................................................ 17 
10.1.2. 
Study dates ..................................................................................................................... 17 
10.1.3. 
Reasons for treatment discontinuation .......................................................................... 17 
10.1.4. 
Reasons for patient’s study discontinuation .................................................................. 19 


 
10.1.5. 
Protocol deviations ........................................................................................................ 19 
11. 
APPENDIX II. DEMOGRAPHIC ANALYSIS AND EFFICACY EVALUATION ............ 20 
11.1. 
BASELINE CHARACTERISTICS ................................................................................................... 20 
11.1.1. 
Patients characteristics at baseline ............................................................................... 20 
11.1.2. 
Histology and time from diagnosis ................................................................................ 20 
11.1.3. 
Prior anticancer therapy................................................................................................ 21 
11.1.4. 
Prior history ................................................................................................................... 22 
11.1.5. 
Physical examination, vital signs, electrocardiogram and other tests ........................... 23 
11.1.6. 
Hematological values at baseline .................................................................................. 24 
11.1.7. 
Biochemical values at baseline ...................................................................................... 24 
11.1.8. 
Other metabolic values at baseline ................................................................................ 25 
11.1.9. 
Signs and symptoms at baseline ..................................................................................... 25 
11.1.10. 
Concomitant therapy ...................................................................................................... 26 
11.2. 
EFFICACY ................................................................................................................................. 27 
11.2.1. 
Response ........................................................................................................................ 27 
11.2.2. 
Time-to-event variables ................................................................................................. 28 
12. 
APPENDIX III. SAFETY EVALUATION .............................................................................. 29 
12.1. 
EXTENT OF EXPOSURE .............................................................................................................. 29 
12.1.1. 
Treatment administration............................................................................................... 29 
12.1.2. 
Dose administration delays............................................................................................ 30 
12.1.3. 
Skipped doses ................................................................................................................. 31 
12.1.4. 
Dose reduction ............................................................................................................... 32 
12.2. 
SAFETY .................................................................................................................................... 33 
12.2.1. 
Adverse events................................................................................................................ 33 
12.2.2. 
Grade 3-4 adverse events............................................................................................... 35 
12.2.3. 
Deaths and serious adverse events (SAEs) .................................................................... 35 
12.3. 
LABORATORY EVALUATION ..................................................................................................... 36 
12.3.1. 
Hematological abnormalities ......................................................................................... 36 
12.3.2. 
Biochemical abnormalities ............................................................................................ 37 
12.3.3. 
Other metabolic abnormalities ...................................................................................... 39 
12.4. 
VITAL SIGNS ............................................................................................................................ 40 
12.5. 
PROPHYLACTIC AND CONCOMITANT MEDICATION DURING TREATMENT .................................. 40 
12.6. 
CARDIAC SAFETY ..................................................................................................................... 41 
12.7. 
OTHER TESTS AND PROCEDURES .............................................................................................. 42 
13. 
APPENDIX IV. FIGURES ........................................................................................................ 43 
14. 
APPENDIX V. DB LISTINGS. ................................................................................................. 47 
15. 
SAP VERSION HISTORY. ....................................................................................................... 48 


 
LIST OF ABBREVIATIONS 
AE(s) 
Adverse Event(s) 
ALT  
Alanine Aminotransferase  
ANC 
Absolute neutrophil count 
AP 
Alkaline Phosphatase  
APTT 
Activated Partial Thromboplastin Time  
AST  
Aspartate Aminotransferase  
ATC 
Anatomical Therapeutic Chemical 
BPD 
Blood Diastolic Pressure 
BPS 
Blood Systolic Pressure 
BSA 
Body Surface Area 
CA 
Calcium 
CI(s) 
Confidence Interval(s) 
CR 
Complete Response 
CrCl 
Creatinine Clearance 
CRF 
Case Report Form  
CTC(s) 
Circulating Tumor Cells 
CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse Events 
DB 
Data Base 
DLT 
Dose-Limiting Toxicity  
DOR 
Duration of Response 
ECG 
Electrocardiogram  
ECOG 
Eastern Cooperative Oncology Group 
EFT 
Ewing Family of Tumors 
GCP 
Good Clinical Practice  
HIV 
Human Immunodeficiency Virus  
ICH 
International Conference on Harmonisation  
IEC/IRB 
Independent Ethics Committee/Institutional Review Board 
IND 
Investigational New Drug 
i.v. 
Intravenous(ly) 
LD 
Longest Diameter  
LDH 
Lactate Dehydrogenase  
LNL 
Lower Normal Limit  
LVEF 
Left Ventricular Ejection Fraction 
MAP 
Mean Arterial Pressure 
MedRA 
Medical Dictionary for Regulatory Activities 
MTD 
Maximum Tolerated Dose  
NCI 
National Cancer Institute 
OR 
Objective Response 
ORR 
Overall Response Rate 
OS 
Overall Survival 
PD 
Progression Disease 
PFS 
Progression-Free Survival 
PGx 
Pharmacogenomic 


 
PK 
Pharmacokinetic(s)  
PNET 
Primitive Neuroectodermal Tumor 
PR 
Partial Response 
PS 
Performance Status 
Q4wk 
Every Four Weeks 
RD 
Recommended Dose 
RECIST  
Response Evaluation Criteria In Solid Tumors 
SAE(s) 
Serious Adverse Event(s) 
SAP 
Statistical Analysis Plan 
SD 
Stable Disease  
TCR 
Tumor Control Rate 
ULN 
Upper Limit of Normal 
v. 
Version 
WHO 
World Health Organization  
WPP 
Worst Per Patient  
 
1. 
Introduction 
1.1. Study rationale 
This Statistical Analysis Plan (SAP) explains in detail the statistical analyses that will be 
carried out for PharmaMar PM104-B-003-10 study. The analyses described in this SAP are 
based upon and supplement those described in the study protocol (dated 30-07-2010). 
1.2. Information on study drug 
PM00104 is a new synthetic alkaloid that has been selected for clinical development based on 
its in vitro activity against human solid and non-solid tumor cell lines, its in vivo activity in 
xenografted human tumors, as well as an acceptable non-clinical toxicology profile. 
2. 
Objectives 
The study protocol states the following: 
2.1. Primary 
 
To determine the antitumor activity of Zalypsis® administered as a 1-hour intravenous (i.v) 
infusion on Day 1, 8 and 15 every four weeks (d1, d8 and d15; q4wk) to patients with 
advanced and/or metastatic Ewing family of tumors (EFT). 
2.2. Secondary 
 
To determine time-to-event efficacy parameters. 
 
To characterize the safety profile and tolerability of Zalypsis® in patients with unresectable 
advanced and/or metastatic EFT. 
 
To characterize the pharmacokinetics (PK) of Zalypsis® when administered as a single-
agent to patients with EFT. 
 
To determine the pharmacodynamic profile by measuring the effect of Zalypsis® on the 
number of Ewing’s sarcoma circulating tumor cells (CTCs) at different times of treatment 
and its correlation with the clinical outcome. 
 
To determine the pharmacogenomic (PGx) profile. Hypothesis-generating exploratory PGx 
analyses will be conducted to correlate the molecular parameters found in the tumor and 
blood samples of the patients with the clinical results achieved with Zalypsis®. 


 
3. 
Study design 
Multicenter, open label, phase II clinical trial with single-agent PM00104 given as a 1-hour i.v. 
infusion on d1, d8 and d15, q4wk, to patients with advanced and/or metastatic EFT who failed 
at least to one line of standard chemotherapy. 
The primary endpoint of the study is the overall response rate (ORR), defined as the percentage 
of patients with objective response (OR), either complete response (CR) or partial response 
(PR), as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. 
4. 
Study populations 
4.1. Analysis populations 
Patients with EFT progressing after at least one line of standard treatment with systemic 
chemotherapy are eligible for this trial. To be included in the study, patients have to fulfill all 
inclusion criteria and none of the exclusion criteria. 
4.1.1. Efficacy population 
To be evaluable for efficacy, eligible patients must have received at least four infusions of the 
six infusions corresponding to the first two cycles and have at least one disease measurement 
recorded not less than six weeks after treatment onset.  
In addition, any eligible patient who receives at least two of the three infusions in one treatment 
cycle and develops disease progression or dies due to progressive disease (PD) prior to 
response evaluation will be considered evaluable for the main endpoint (ORR) and will be 
categorized as an “early progression”.  
Patients withdrawn due to toxicity without any tumor assessment after the start of study 
treatment will be considered as “treatment failures” and will not be replaced. 
Patients withdrawn due to significant clinical deterioration of unknown reason, or due to 
hypersensitivity reactions or unrelated AEs, and patients who refuse to continue on study for 
any reason without any tumor assessment after the start of study treatment will be considered 
not evaluable for efficacy and will be replaced. 
4.1.2. Safety population 
All patients who have received at least one total or partial infusion of Zalypsis® will be 
included in the safety analysis. 
5. 
Endpoints 
5.1. Primary endpoints 
 
Overall response rate (ORR): is defined as the percentage of patients with confirmed OR, 
either CR or PR response according to the RECIST v1.1. 
5.2. Secondary endpoints 
 
Duration of response (DOR): is defined as the time between the date when the response 
criteria (PR or CR, whichever is reached first) are fulfilled and the first date when disease 
progression, recurrence or death is objectively documented (taking the smallest 
measurements documented since the treatment started as reference for PD). DOR censoring 
rules are the same as for progression-free survival (PFS) and are described below. 
 
Progression-free survival (PFS): is defined as the time from the date of the first study drug 
administration to the date of documented progressive disease (PD) by RECIST v1.1 or 
death (regardless of the cause of death). If the patient receives further antitumor therapy 
before PD and within the timeframe expected for first follow-up, PFS will be censored on 
the date of the last disease assessment prior to the administration of this antitumor therapy. 
If the patient is lost to follow-up for the assessment of progression, or has more than one 


 
missing follow-up between the date of last tumor assessment and the date of progression, 
death or further antitumor therapy, the PFS will be censored at the date of last valid tumor 
assessment before the missing evaluations. PFS at three and PFS at six months (PFS3 and 
PFS6) are defined as the Kaplan Meier estimates of PFS at these time points.  
 
Overall survival (OS): is defined as the time from the first day of study treatment 
administration to the date of death (or the last date when the patient is known to be alive). 
Survival will be followed every three months until death, or until the date of study 
termination, whichever occurs first. OS at 12 months (OS12) is defined as the Kaplan-
Meier estimates of OS at this time point.  
 
Safety profile: adverse events (AEs), serious adverse events (SAEs), laboratory evaluations, 
deaths and the reason for study discontinuations, dose delays, modifications or omissions 
will be analyzed. All AEs and SAEs will be classified according to the NCI-CTCAE, v4.0, 
and will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). 
5.3. Other endpoints 
 
Treatment exposure:  
- 
Date of treatment start: Date of treatment administration in Cycle 1. 
- 
Date of last dose: Date of last treatment administration in the last cycle. 
- 
End of treatment: Day of the last study drug administration plus 30 days, or onset 
of subsequent therapy or death, whichever occurs first. 
- 
Time on treatment: Time from treatment start to the “end of treatment” date. 
- 
A cycle: Period of time (planned as 4 weeks plus the potential administration 
delays) between two dose infusions. The duration of the last cycle (for the purpose 
of exposure calculations) will be 4 weeks. Therefore, the exposure duration will be 
calculated as the time between the day of the first dose and the day of last dose + 4 
weeks (28 days) (expressed in weeks). 
- 
Body Surface Area (BSA): The calculated surface of a human body (expressed in 
m²). BSA will be calculated using the Dubois & Dubois formula: 
725
.0
425
.0
2
)
(
)
(
007184
.0
)
(
cm
height
kg
weight
m
BSA



 
 
- 
Total cumulative dose: The sum of all the study drug doses from the first cycle 
until last cycle, including the dose received in last cycle (expressed in mg/m²). 
- 
Absolute dose intensity: The actual cumulative dose divided by number of weeks of 
exposure (expressed in mg/m²/wk). 
- 
Intended dose intensity: The planned dose per cycle divided by the planned weeks 
by cycle (expressed in mg/m²/wk). 
- 
Relative dose intensity: The ratio of absolute dose intensity (x100) divided by the 
intended dose intensity (expressed in percentage). 
 
Pharmacokinetic (PK) and pharmacodinamic profile.  
- 
These analyses will be described in different documents not included in this SAP. 
For further details please refer to the study Protocol. 
 
Pharmacogenomic (PGx) profile. 
- 
The PGx analysis will be described in a different document not included in this 
SAP. For further details please refers to the study Protocol. 


 
6. 
Sample size  
6.1. Sample size determination 
In this phase II trial, an optimum two-stage design to test the null hypothesis that the ORR is ≤ 
3% versus the alternative that ORR ≥ 20% was selected. After testing the drug on 12 patients in 
the first stage, the trial will be terminated if there is not any responder. If the trial goes on to the 
second stage, a total of 29 patients will be studied, 12 from the first stage and 17 from the 
second stage. If the total number responding is less than or equal to 2 (two patients), the drug 
will be considered not interesting in the setting of patients treated with this disease and with this 
schedule.  
This design has an expected sample size of 17.20 and a probability of early termination of 0.69 
under the null hypothesis. If the drug is actually not effective, there is a 0.05 probability of 
concluding that it is (type I error). If the drug is actually effective, there is a 0.1 probability of 
concluding that it is not (type II error). 
7. 
Statistical analysis 
7.1. Treatment discontinuation and protocol deviations management 
The study populations will be summarized and listed in Section 10 (Appendix I), including the 
number of patients in the following populations: 
 
Eligible patients. 
 
Included patients. 
 
All treated patients (safety population). 
 
Patients evaluable for the main endpoint (efficacy population). 
Reasons for not belonging to any population will be detailed. 
The accrual and study discontinuation details will be presented descriptively. The reasons for 
treatment discontinuation will be described by counts and percentages, overall and by number 
of cycles received. Reasons of treatment discontinuation other than disease progression will be 
detailed.  
Reason for patient’s study withdrawal will be also detailed. These may include: 
 
Study termination 
 
Patient refusal 
 
Investigator decision 
 
Lost to follow up 
 
Death 
 
Never treated 
 
Other reasons 
A protocol deviation is defined as any departure from what is described in the protocol of a 
clinical trial approved by an Independent Ethics Committee/Institutional Review Board 
(IEC/IRB) and Competent Authorities. Therefore, it applies to deviations related to patient 
inclusion and clinical procedures (e.g., assessments to be conducted or parameters to be 
determined), and also to other procedures described in the protocol that concern the Good 
Clinical Practice (GCP) guidelines or ethical issues (e.g., issues related to obtaining the 
patients’ Informed Consent, data reporting, the responsibilities of the Investigator, etc.). 
Protocol deviations will be summarized for all patients. A summary table with the number of 
patients with inclusion/exclusion deviations will be presented per criterion. These patients will 
be listed with the criteria that are not met. Deviations with no effects on the risk/benefit ratio of 
the clinical trial will be distinguished from those that might have an effect on this risk/benefit 
ratio. 


 
A study of the protocol deviations deemed relevant according to the Data Management Plan 
and Oncology review will be made following the International Conference on Harmonisation 
(ICH) guidelines. The protocol deviations will be listed by type of deviation, including (but not 
restricted to) the following categories: 
 
Patients not satisfying inclusion / exclusion criteria. 
 
Patients developing withdrawal criteria but not withdrawn. 
 
Patients receiving incorrect dose or at an incorrect schedule. 
 
Patients receiving forbidden concomitant treatment. 
 
Patients receiving treatment at the same dose and schedule but not satisfying criteria for 
treatment continuation. 
7.2. Demographic analysis 
Demographics and baseline characteristics will be summarized for all recruited patients 
following the standard tables detailed in Section 11 (Appendix II).  
Continuous variables will be summarized and presented with summary statistics, i.e., median 
and range, mean, standard deviation and categorical variables will be summarized in frequency 
tables. 
 
In case of pre-treatment characteristics with multiple measurements per patient before the start 
of treatment (laboratory assessments, vital signs) the baseline measurement will be considered 
the last value prior to or on the first day of treatment. 
Age, sex, baseline weight, height, BSA, Eastern Cooperative Oncology Group performance 
status (ECOG-PS), vital signs (heart rate, blood pressure and body temperature), left ventricular 
ejection fraction (LVEF), electrocardiogram (ECG), signs and symptoms and concomitant 
medication results will be summarized descriptively. Age is calculated based on the date of 
birth at the time date of registration date. 
Age will be calculated based on the date of birth and the date of informed consent and rounded 
to integer numbers (by defect). 
 
25
.
365
birth)
 
of
 
(date
-
consent)
 
informed
 
of
 
date
(

Age
 
 
Tumor characteristics: histology diagnosis, primary site, time from diagnosis, stage, number of 
organs involved and sites of disease, time from last documentation of PD before start of 
treatment with Zalypsis® will be described. 
A frequency tabulation of the number of patients with the different types of previous cancer 
surgery, radiotherapy and systemic anticancer therapy will be given. 
A summary of prior relevant history and signs and symptoms will be presented per patient. 
Laboratory values at baseline will be tabulated. Median values, ranges and NCI-CTCAE grades 
will be displayed by laboratory parameters. 
7.3. Statistical methodology for efficacy 
The main efficacy analysis will be performed in the efficacy population. A sensitivity analysis 
of efficacy results will be done in the population of all treated patients (safety population). 
Binomial estimates with exact 95% confidence intervals (CIs) will be calculated for the 
analysis of the main endpoint (ORR). Time-to-event endpoints (DOR, PFS3, PFS6 rates and 
OS rate at 12 months) will be analyzed according to the Kaplan-Meier method.  


 
If relevant, efficacy parameters versus baseline covariates will be analyzed and appropriate test 
will be used (i.e., the Fisher exact test for categorical variables, the log-rank test or Cox 
regression for time-to-event variables, etc.).  
Graphical representations of tumor shrinkage will be represented by waterfall plots or spider 
plots if applicable. 
Statistical tests, if and when they are carried out, will only have an exploratory purpose and 
have a threshold of  = 5%. 
See Section 11 (Appendix II) for further details on the efficacy analysis and Section 13 
(Appendix IV) for graphic templates. 
7.4. Statistical methodology for safety  
Descriptive statistics, tabulation and graphic representation will be used for the evaluation of 
safety for this phase II study, as described in Section 12 (Appendix III) and Section 13 
(Appendix IV).  
7.4.1. Exposure 
Cumulative dose, dose intensity and relative dose intensity, cycle delay, and dose modifications 
or omissions will be described following standard tables detailed in Section 12.1 (Appendix 
III). 
7.4.2. Cycle delays, skipped doses and dose modifications 
Patients who receive only one infusion of Zalypsis® and have treatment discontinuation within 
the first cycle due to any reason different from drug related adverse event (eg: disease 
progression, patient’s refusal), will not be considered for the calculations of the number of dose 
administration delays, doses skipped or dose reductions.   
Dose delay is defined by a delay in the administration of the first infusion of one cycle (Day 1 
infusion). Infusions not administered on Days 8 and/or 15 of the cycle are considered skipped 
doses and are analyzed separately.  
The item "Dose delayed: No/Yes" in the case report form (CRF) will be used to calculate the 
delay. For doses considered as delayed by the investigator, the delay will be calculated as: 
Administration delay (in days) will be equal to the date of start of the actual cycle minus the 
date of start of previous cycle minus 28 days (planed cycle duration, in days). 
A second analysis taking into account the drug administration dates rather than the item “dose 
delay” completed by the investigator will be performed in order to know the calculated delay in 
days. 
The first cycle is excluded from all calculation of cycle delay (and the denominator used for 
calculations will be equal to the number of cycles susceptible to be delayed). 
The distribution of delays will be studied by means of counts and percentages (the denominator 
used for calculations will be equal to the number of patients/cycles susceptible of delay). The 
reasons for infusion delay will be detailed, specifying how many were due to treatment and 
how many were not. Within delays attributable to treatment, hematological and non-
hematological reasons will be outlined, and the specific reasons will be detailed (administrative 
reasons will be analyzed separately and additional tables will be prepared after exclusion of 
those delays). 
Skipped dose or dose omission is defined as every infusion not administered on Day 8 and/or 
15 of the cycle. The distribution of skipped doses will be studied by means of counts and 
percentages (the denominator used for calculations will be equal to the number of 
patients/cycles susceptible to have an omission) and a detailed listing of patients with skipped 


 
dose and reason will be showed. Also reasons for dose omission will be detailed, specifying 
how many were due to treatment and how many were not.  
Dose reduction refers to any dose modification occurred after the first drug administration. The 
distribution of reductions will be studied by means of counts and percentages (the denominator 
used for calculations will be equal to the number of patients/cycles susceptible to be reduced). 
All dose reductions will be considered and described (per cycle and patient), specifying the 
magnitude and the reason for reduction (hematological toxicity, non-hematological toxicity or 
other causes not due to treatment). 
7.4.3. Adverse events 
Adverse events (AEs) will be graded according to NCI-CTCAE v4.0 and will be coded using 
the MeDRA, v11.0. The incidence and grade of AEs and laboratory abnormalities will be 
calculated considering the most severe grade per patient and cycle. The numeric form of the 
NCI-CTCAE grade (Grade 0, 1, 2, 3, 4, 5) will be used for all tables and listings. Grades 
displayed in tables and figures will be grouped in categories to simplify reading (occurrence of 
grade 5 of the NCI-CTCAE will be in tables indicated by Grade 4* and the asterisk (*) will 
have associated with a footnote where further detail information is provided). Totals will be 
provided in all tables. 
As far as all the events and abnormalities are concerned, the NCI-CTCAE grade will be used 
wherever an NCI-CTCAE grading exists. Otherwise, the severity will be noted. As a 
convention, the term "Grade" will always be used. Toxicities will be described according to the 
worst NCI-CTCAE grade reported or, for toxicities which do not form the subject of NCI-
CTCAE classification, according to the worst severity. 
Descriptive statistics will be used to present the profiles of drug-related AEs, drug-related 
deaths, SAEs and drug-related treatment discontinuations and the observed grade 1-4 toxicities, 
per patient and per cycle. The incidence and grade of AEs and laboratory abnormalities will be 
calculated considering the most severe grade per patient and infusion and will be displayed in 
frequency tables using counts and percentages. 
7.4.4. Deaths and serious adverse events 
Database listings of deaths and serious adverse events will be provided, including at least date 
of onset and resolution (if applicable), severity, relationship to study drug, significant 
consequences and action taken. Deaths on treatment (within 30 days from last study drug 
administration) will also be included. 
7.4.5. Laboratory evaluations 
Hematology 
Hematological abnormalities classified according to the NCI-CTCAE will be calculated in all 
cycles. The worst grade reached by each patient during treatment will be also calculated. 
If serious abnormalities occur, special follow-up including calculation of nadir values and 
median time to recover to baseline values with 95% confidence intervals (CIs) and descriptive 
tables will be made to find out the pattern of thrombocytopenia and neutropenia within and 
between the different cycles. 
If appropriate, these tables might be complemented with boxplots for the nadir of neutrophils 
and platelets count by cycle along the treatment. Furthermore, graphs of the inter-cycle time 
course of neutropenia and thrombocytopenia will be provided. Graphs comparing the time 
course during the first and second or further cycles will be created, if required. 
Serum Biochemistry 
The non-hematological laboratory abnormalities (i.e., NCI-CTCAE grades) of transaminases, 
creatinine, creatine phosphokinase (CPK), bilirubin, alkaline phosphatase (AP), etc., by patient 


 
and cycle, will be calculated as explained for hematological disorders. The worst grade reported 
for each patient during treatment be recorded. 
If appropriate, the time to onset and recovery from selected non-hematological abnormalities 
(i.e., hepatic enzymes) will be illustrated graphically and by means of descriptive statistics. 
 
Shift from baseline 
The shift of severity grades from baseline to the worst occurrence during treatment will be 
tabulated. The time to onset and recovery from neutrophils, platelets, and hepatic enzymes 
elevations will be illustrated by means of descriptive statistics. 
7.4.6. Other safety evaluations 
Physical examination evaluation (normal/abnormal) and vital signs at baseline and during 
treatment will be described by means of frequency counts and summary statistics. 
Electrocardiogram (ECG) evaluation (normal/abnormal) will be summarized with frequency 
counts. 
Continuous variables (LVEF and LVEF Normal Range) will be summarized and presented with 
summary statistics, i.e., mean, standard deviation, median and range.  
Tabulation of baseline values and evolution during treatment will be presented for PS, weight, 
and LVEF, troponin I measurement. 
7.4.7. Concomitant Medication 
Concomitant therapies will be categorized per the World Health Organization Anatomical 
Therapeutic Chemical (WHO-ATC) class and coded term. The number of patients receiving 
each type of therapy during the treatment phase will be tabulated in two separate tables: a 
frequency tabulation of the different therapies that started pre-study, and a frequency tabulation 
of the different therapies that started during the study. Each table will be generated by system, 
indication and agent family, WHO-ATC levels 1, 2 and 4 and once by coded term. The 
accompanying listing will contain all concomitant therapies. 
See Section 12.2-12.5 (Appendix III) for further details on the safety analysis. 
7.5. Pharmacokinetic (PK), Pharmacodynamic profile and pharmacogenomic (PGx) 
evaluation 
These analyses will be described in two different documents not included in this SAP.  
8. 
Other statistical analysis considerations 
8.1. Missing value management 
In case of missing values in the determination of protocol deviations (for instance, time from 
last anticancer treatment to start of treatment with Zalypsis®) the most conservative approach 
will be taken for the evaluation. 
The cycles with missing information regarding laboratory values or adverse events will be 
subtracted from the denominator of the tables. 
8.1.1. Imputation in incomplete dates  
Before registration  
If the day of a date is unknown (i.e, UK/Jan/09) then the imputed day will be the 15th. If the 
month is also unknown (i.e, UK/UK/09) then the imputed date will be July 1st. This assumption 
will be valid if the imputed date is earlier than the informed consent's date. Otherwise the 
imputed date will be the first day of the informed consent's month date (i.e., 01 / informed 
consent’s month / year).  


 
After end of treatment  
To ensure the most conservative approach for the main time-to-event variables (i.e., PFS and 
OS) that can be affected by missing values, the following rules will be implemented: if the day 
of a date is unknown then the imputed day will be the 1st; if the month is also unknown, then 
the imputed date will be July 1st. This assumption will be valid if the imputed date occurs later 
than the last drug administration date; otherwise the imputed date will be either last drug 
administration date plus the planed cycle duration (28 days), or the last day of the reported 
month, whichever occurs first. 
8.2. Data analysis conventions 
Percentages in the summary tables will be rounded to one decimal unless otherwise specified, 
and, therefore, may not always add up to exactly 100%. 
 
Calcium can be falsely low if hypoalbuminemia is present. Serum album is <4.0 g/dl, 
hypocalcemia will be reported after the following correction is performed:  
 
Calcium corrected (mMol/l) = Calcium (mMol/l) + 0.2 x (4 – Albumin(g/dl)) 
 
Calcium corrected (g/dl) = Calcium (g/dl) + 0.8 x (4 – Albumin(g/dl)) 
 
In case of albumin and calcium results are not performed in the same date then the nearest 
albumin result to the calcium will be taken and if a calcium result falls at an equal time between 
two albumin results, then the earlier albumin result will be used for the calculation. 
If there is no any albumin results or the result exceeds more than 30 days from calcium result 
then no correction will be used. 
8.3. Multivariate analyses 
If appropriate, exploratory Cox regression for multivariate analysis of main covariate effects 
will be used for time-dependent efficacy parameters, and logistic regression for the evaluation 
of covariates associated with best overall response. 
8.4. Fixed, random or mixed effects models 
Not applicable. 
8.5. Subgroup Analysis 
Not applicable. 
8.6. Interim Analysis 
A futility analysis is planned when 12 patients evaluable for efficacy are recruited according to 
the Simon’s design. Other non-scheduled analyses might be performed exclusively for 
enhancing patient’s safety. 
8.7. Independent review committee 
Because of safety reasons, an independent cardiologist will undertake a review of all the ECGs 
and echocardiograms performed to the patients during the course of the study, in order to 
evaluate the cardiotoxicity risk of PM00104. For this purpose, an independent cardiologist´s 
report not included in this SAP will be presented as an Appendix of the CSR. 
9. 
Statistical software 
Oracle Clinical will be used for double data entry and clinical data management.  
SAS v9.2 will be used for all the statistical analysis. 


 
 
TABLES, LISTINGS AND FIGURE SHELLS 
 
NOTE:  Each particular table, listing and figure will have a comprehensive header and/or 
footnotes. If the number of items would not yield appropriate tabular or graphic representations, 
detailed listings will be shown instead. 
 
TABLE 10.1.1.1  PATIENTS DISPOSITION BY INSTITUTION. ...................................................................... 17 
TABLE 10.1.1.2  LISTING OF PATIENTS NOT ELIGIBLE. ............................................................................ 17 
TABLE 10.1.1.3  LISTING OF PATIENTS INCLUDED BUT NOT TREATED. .................................................... 17 
TABLE 10.1.1.4  LISTING OF PATIENTS NOT EVALUABLE FOR SAFETY. .................................................... 17 
TABLE 10.1.1.5  LISTING OF PATIENTS NOT EVALUABLE FOR EFFICACY. ................................................ 17 
TABLE 10.1.2.1  STUDY DATES. .............................................................................................................. 17 
TABLE 10.1.3.1  REASONS FOR TREATMENT DISCONTINUATION. ............................................................ 18 
TABLE 10.1.3.2  REASONS FOR TREATMENT DISCONTINUATION BY NUMBER OF CYCLES RECEIVED. ...... 18 
TABLE 10.1.3.3  TIME ON TREATMENT (MONTHS)................................................................................... 18 
TABLE 10.1.3.4  LISTINGS OF PATIENTS AND TIME ON TREATMENT. ....................................................... 18 
TABLE 10.1.3.5  LISTINGS OF TREATMENT DISCONTINUATION OTHER THAN PROGRESSIVE DISEASE. ..... 18 
TABLE 10.1.3.6  LISTING OF TREATMENT DISCONTINUATION DUE TO ADVERSE EVENTS (AES). ............. 18 
TABLE 10.1.3.7  LISTING OF TREATMENT DISCONTINUATION DUE TO DEATH. ........................................ 19 
TABLE 10.1.4.1  REASON FOR STUDY DISCONTINUATION. ...................................................................... 19 
TABLE 10.1.4.2  LISTING OF OTHER REASONS STUDY DISCONTINUATION. .............................................. 19 
TABLE 10.1.5.1  ELIGIBILITY: PATIENTS WHO ENTERED THE STUDY EVEN THOUGH THEY DID NOT 
SATISFY THE ENTRY CRITERIA. ............................................................................................................... 19 
TABLE 11.1.1.1  BASELINE CHARACTERISTICS: SUMMARY STATISTICS. ................................................. 20 
TABLE 11.1.1.2  SUMMARY STATISTICS: BASELINE CHARACTERISTICS. .................................................. 20 
TABLE 11.1.2.1  TUMOR HISTOLOGY. ..................................................................................................... 20 
TABLE 11.1.2.2  SITES FOR TARGET AND NON-TARGET LESIONS. ............................................................ 21 
TABLE 11.1.2.3  LISTING OF PATIENT HISTOLOGY. ................................................................................. 21 
TABLE 11.1.2.4  LISTING OF TARGET AND NON TARGET LESIONS............................................................ 21 
TABLE 11.1.2.5 SUMMARY STATISTICS OF CANCER DIAGNOSIS CHARACTERISTICS: TIME FROM 
DIAGNOSIS AND TIME FROM LAST PROGRESSION DISEASE (PD) TO FIRST INFUSION OF STUDY DRUG. ..... 21 
TABLE 11.1.3.1  SURGERY (PALLIATIVE + CURATIVE). ........................................................................... 21 
TABLE 11.1.3.2  RADIOTHERAPY. ........................................................................................................... 21 
TABLE 11.1.3.3  PATIENTS WITH PRIOR SURGERY. .................................................................................. 21 
TABLE 11.1.3.4  SUMMARY STATISTICS OF PRIOR SYSTEMIC ANTICANCER THERAPY. ............................. 22 
TABLE 11.1.3.5  NUMBER OF LINES AND AGENTS OF SYSTEMIC ANTICANCER THERAPY. ........................ 22 
TABLE 11.1.3.6  SUMMARY OF LINES AND AGENTS OF SYSTEMIC ANTICANCER THERAPY....................... 22 
TABLE 11.1.4.1  PRIOR HISTORY. ............................................................................................................ 22 
TABLE 11.1.5.1  PHYSICAL EXAMINATION AT BASELINE......................................................................... 23 
TABLE 11.1.5.2  LISTINGS OF PATIENTS WITH POSITIVE PREGNANCY TEST. ............................................ 23 
TABLE 11.1.5.3  LISTINGS OF PATIENTS WITH NO ADEQUATE CONTRACEPTION. ..................................... 23 
TABLE 11.1.5.4   LISTING OF ECOG PERFORMANCE STATUS. ................................................................. 23 
TABLE 11.1.5.5  LISTING OF PATIENTS WITH ABNORMAL ECG AT BASELINE. ......................................... 23 
TABLE 11.1.5.6  LISTING OF PATIENTS WITH LVEF LOWER THAN THE NORMAL RANGE. ........................ 23 
TABLE 11.1.5.7  SUMMARY STATISTICS: PHYSICAL EXAMINATION AND VITAL SIGNS. ............................ 23 
TABLE 11.1.6.1  HEMATOLOGICAL ABNORMALITIES AT BASELINE. ........................................................ 24 
TABLE 11.1.6.2  HEMATOLOGY AT BASELINE. LAST VALUE BEFORE TREATMENT. ................................. 24 
TABLE 11.1.6.3  HEMATOLOGY AT BASELINE: PATIENTS WITH HEMATOLOGICAL PARAMETERS MISSING.
 ............................................................................................................................................................... 24 
TABLE 11.1.6.4  HEMATOLOGY AT BASELINE. ABNORMALITIES GRADE ≥ 2. .......................................... 24 
TABLE 11.1.7.1  BIOCHEMICAL ABNORMALITIES AT BASELINE............................................................... 24 
TABLE 11.1.7.2  BIOCHEMISTRY AT BASELINE. LAST VALUE BEFORE TREATMENT. ................................ 24 
TABLE 11.1.7.3  BIOCHEMISTRY AT BASELINE. PATIENTS WITH MISSING BIOCHEMICAL PARAMETERS. .. 25 
TABLE 11.1.7.4  BIOCHEMISTRY AT BASELINE. ABNORMALITIES GRADE ≥ 2. ......................................... 25 
TABLE 11.1.8.1  OTHER METABOLIC ABNORMALITIES AT BASELINE (HYPER-HYPO). .............................. 25 
TABLE 11.1.8.2  OTHER METABOLIC ABNORMALITIES AT BASELINE. LAST VALUE BEFORE TREATMENT.
 ............................................................................................................................................................... 25 
TABLE 11.1.8.3  PATIENTS WITH MISSING METABOLIC PARAMETERS. ..................................................... 25 
TABLE 11.1.8.4  OTHER METABOLIC AT BASELINE. ABNORMALITIES GRADE ≥ 2. ................................... 25 


 
 
TABLE 11.1.9.1  SUMMARY STATISTICS: SIGNS AND SYMPTOMS. ........................................................... 25 
TABLE 11.1.9.2  MEDIAN OF SIGNS AND SYMPTOMS ............................................................................... 26 
TABLE 11.1.9.3  LISTING OF BASELINE CHARACTERISTICS: SIGNS AND SYMPTOMS (GRADE ≥ 2). .......... 26 
TABLE 11.1.10.1  AGENTS OF CONCOMITANT THERAPY STARTED AT BASELINE. .................................... 26 
TABLE 11.1.10.2  CONCOMITANT MEDICATION CHARACTERISTICS STARTED AT BASELINE. ................... 26 
TABLE 11.1.10.3  CONCOMITANT THERAPY STARTED AT BASELINE. SUMMARY OF NUMBER, INDICATIONS 
AND SYSTEMS INVOLVED. ....................................................................................................................... 27 
TABLE 11.2.1 1  PATIENTS EVALUABLE FOR EFFICACY. .......................................................................... 27 
TABLE 11.2.1.2  LISTING OF PATIENTS EXCLUDED FROM THE EFFICACY ANALYSIS. ............................... 27 
TABLE 11.2.1.3  OVERALL RESPONSE RATE. ........................................................................................... 27 
TABLE 11.2.1.4  BEST RESPONSE IN ALL EVALUABLE PATIENTS. ............................................................ 27 
TABLE 11.2.1.5  BEST RESPONSE IN ALL TREATED PATIENTS. ................................................................. 28 
TABLE 11.2.1.6  CHARACTERISTICS OF RESPONDERS .............................................................................. 28 
TABLE 11.2.2.1  DURATION OF RESPONSE. ............................................................................................. 28 
TABLE 11.2.2.2  PROGRESSION-FREE SURVIVAL (KAPLAN-MEIER ESTIMATES IN ALL EVALUABLE 
PATIENTS). .............................................................................................................................................. 28 
TABLE 11.2.2.3  OVERALL SURVIVAL. (KAPLAN-MEIER ESTIMATES IN ALL EVALUABLE PATIENTS). ..... 28 
TABLE 11.2.2.4  PROGRESSION-FREE SURVIVAL (KAPLAN-MEIER ESTIMATES IN ALL TREATED 
PATIENTS). .............................................................................................................................................. 28 
TABLE 11.2.2.5  OVERALL SURVIVAL. (KAPLAN-MEIER ESTIMATES IN ALL TREATED PATIENTS). .......... 29 
TABLE 11.2.2.6  FOLLOW-UP PERIOD. ..................................................................................................... 29 
TABLE 12.1.1.1  CYCLES ADMINISTERED. ............................................................................................... 29 
TABLE 12.1.1.2  SUMMARY OF CYCLES ADMINISTERED. ......................................................................... 29 
TABLE 12.1.1.3  DOSE INTENSITY. .......................................................................................................... 29 
TABLE 12.1.2.1  DOSE DELAY ................................................................................................................. 30 
TABLE 12.1.2.2  DOSE DELAY ACCORDING TO THEIR RELATION TO STUDY DRUG. .................................. 30 
TABLE 12.1.2.3  LENGTH OF DOSE DELAY. ............................................................................................. 30 
TABLE 12.1.2.4  DOSE DELAY AND REASON BY CYCLE. .......................................................................... 31 
TABLE 12.1.2.5  LISTING OF PATIENT WITH DOSE DELAY. ....................................................................... 31 
TABLE 12.1.2.6  LISTING OF PATIENTS WITH DOSE INTERRUPTION AND/OR RE-ADMINISTRATION ........... 31 
TABLE 12.1.3.1  SKIPPED DOSES. ............................................................................................................ 31 
TABLE 12.1.3.2  LISTING OF PATIENTS WITH SKIPPED DOSES. ................................................................. 32 
TABLE 12.1.4.1  DOSE REDUCTION. ........................................................................................................ 32 
TABLE 12.1.4.2  DOSE REDUCTION ACCORDING TO THEIR RELATION TO STUDY DRUG. .......................... 32 
TABLE 12.1.4.3  DOSE REDUCTION AND REASON BY CYCLE. .................................................................. 33 
TABLE 12.1.4.4  PATIENTS WITH DOSE REDUCTION. ............................................................................... 33 
TABLE 12.1.4.5  PATIENTS WITH DOSE DELAY AND REDUCTION. ............................................................ 33 
TABLE 12.2.1.1  SUMMARY OF ADVERSE EVENTS. .................................................................................. 33 
TABLE 12.2.1.2  SUMMARY OF HEMATOLOGICAL LABORATORY ABNORMALITIES. ................................. 34 
TABLE 12.2.1.3  SUMMARY OF NON-HEMATOLOGICAL LABORATORY ABNORMALITIES. ......................... 34 
TABLE 12.2.1.4  DRUG-RELATED ADVERSE EVENTS. WORST CASE PER PATIENT .................................... 34 
TABLE 12.2.1.5  DRUG-RELATED ADVERSE EVENTS OCCURRING IN > 10% WORST CASE PER PATIENT. . 34 
TABLE 12.2.1.6  ALL ADVERSE EVENTS REGARDLESS RELATIONSHIP. WORST CASE PER PATIENT. ......... 34 
TABLE 12.2.1.7  ALL ADVERSE EVENTS REGARDLESS RELATIONSHIP OCCURRING IN > 10% WORST CASE 
PER PATIENT. .......................................................................................................................................... 34 
TABLE 12.2.1.8  DRUG-RELATED ADVERSE EVENTS. WORST CASE PER CYCLE. ...................................... 34 
TABLE 12.2.1.9  DRUG-RELATED ADVERSE EVENTS OCCURRING IN > 10% WORST CASE PER CYCLE. .... 34 
TABLE 12.2.1.10  ALL ADVERSE EVENTS REGARDLESS RELATIONSHIP. WORST CASE PER CYCLE. .......... 34 
TABLE 12.2.1.11  ALL ADVERSE EVENTS REGARDLESS RELATIONSHIP OCCURRING IN > 10% WORST 
CASE PER CYCLE. .................................................................................................................................... 34 
TABLE 12.2.1.12  LISTING OF AES LEADING TO DEATH. ......................................................................... 34 
TABLE 12.2.1.13  LISTING OF AES TREATMENT RELATED LEADING TO DEATH. ...................................... 34 
TABLE 12.2.1.14  LISTING OF AES LEADING TO DOSE DELAY. ................................................................ 34 
TABLE 12.2.1.15  LISTING OF AES LEADING TO DOSE REDUCTION. ........................................................ 34 
TABLE 12.2.1.16  LISTING OF AES LEADING TO DOSE DELAY AND REDUCTION. ..................................... 34 
TABLE 12.2.2.1  LISTING OF PATIENTS WITH GRADE 3-4 AES (TREATMENT-RELATED). ......................... 35 
TABLE 12.2.2.2  LISTING OF PATIENTS WITH GRADE 3-4 AES (TREATMENT-RELATED). ......................... 35 
TABLE 12.2.3.1  LISTING OF SAES. ........................................................................................................ 35 
TABLE 12.2.3.2  LISTING OF ALL DEATHS. .............................................................................................. 35 


 
 
TABLE 12.3.1.1  HEMATOLOGICAL ABNORMALITIES DURING TREATMENT, WORST GRADE PER PATIENT.
 ............................................................................................................................................................... 36 
TABLE 12.3.1.2  HEMATOLOGICAL ABNORMALITIES DURING TREATMENT, WORST GRADE PER CYCLE. .. 36 
TABLE 12.3.1.3  PATIENTS WITH ANY HEMATOLOGICAL ABNORMALITIES GRADE 3-4. ........................... 36 
TABLE 12.3.1.4  LISTING OF PATIENTS AND CYCLES WITH HEMATOLOGICAL EVALUATIONS MISSING. ... 36 
TABLE 12.3.1.5  TIME COURSE AND RECOVERY FROM HEMATOLOGICAL TOXICITIES.............................. 37 
TABLE 12.3.1.6  BASELINE GRADE AND EVOLUTION OF HEMATOLOGICAL TOXICITIES. .......................... 37 
TABLE 12.3.2.1  BIOCHEMICAL ABNORMALITIES DURING TREATMENT, WORST GRADE PER PATIENT. .... 37 
TABLE 12.3.2.2  BIOCHEMICAL ABNORMALITIES DURING TREATMENT, WORST GRADE PER CYCLE. ....... 38 
TABLE 12.3.2.3  SUPPORTIVE LISTING: PATIENTS WITH ANY BIOCHEMICAL ABNORMALITIES GRADE 3-4.
 ............................................................................................................................................................... 38 
TABLE 12.3.2.4  LISTING OF PATIENTS AND CYCLES WITH BIOCHEMICAL EVALUATIONS MISSING. ......... 38 
TABLE 12.3.2.5  TIME COURSE AND RECOVERY FROM BIOCHEMICAL TOXICITIES. .................................. 38 
TABLE 12.3.2.6  BASELINE GRADE AND EVOLUTION OF BIOCHEMICAL TOXICITIES. ................................ 39 
TABLE 12.3.3.1  METABOLIC ABNORMALITIES DURING TREATMENT, WORST GRADE PER PATIENT ......... 39 
TABLE 12.3.3.2  METABOLIC ABNORMALITIES DURING TREATMENT, WORST GRADE PER CYCLE. .......... 39 
TABLE 12.3.3.3  METABOLIC ABNORMALITIES GRADE PER CYCLE.......................................................... 39 
TABLE 12.3.3.4  LISTING OF PATIENTS AND CYCLES WITH METABOLIC EVALUATIONS MISSING. ............ 39 
TABLE 12.4.1 VITAL SIGNS. .................................................................................................................... 40 
TABLE 12.5.1  PATIENTS WHO RECEIVED PROPHYLACTIC MEDICATION. ................................................. 40 
TABLE 12.5.2  PROPHYLACTIC THERAPY BY PATIENT. ............................................................................ 40 
TABLE 12.5.3  AGENTS OF CONCOMITANT THERAPY STARTED DURING TREATMENT. ............................. 40 
TABLE 12.5.4  CONCOMITANT MEDICATION CHARACTERISTICS DURING TREATMENT. ........................... 40 
TABLE 12.5.5  SUMMARY OF CONCOMITANT MEDICATION DURING TREATMENT. ................................... 41 
TABLE 12.6.1  LISTING OF PATIENTS WITH CARDIAC AES AND CHARACTERISTICS. ................................ 41 
TABLE 12.6.2  LISTING OF ECG EVOLUTION PER PATIENT. ..................................................................... 41 
TABLE 12.6.3  LISTING OF PATIENTS WITH ABNORMAL ECG. ................................................................. 41 
TABLE 12.6.4  MEDIAN CHANGE IN LVEF (%). ...................................................................................... 41 
TABLE 12.6.5  LISTING OF LVEF/ECG EVOLUTION PER PATIENT. .......................................................... 41 
TABLE 12.6.6  LVEF CHARACTERISTICS PER PATIENT. ........................................................................... 42 
TABLE 12.6.7  MEDIAN CHANGE IN TROPONIN I. .................................................................................... 42 
TABLE 12.6.8  LISTING OF TROPONIN I EVOLUTION. ............................................................................... 42 
TABLE 12.6.9  TROPONIN I CHARACTERISTICS PER PATIENT. .................................................................. 42 
TABLE 12.7.1.1 OTHER TESTS AND PROCEDURES. .................................................................................. 42 
FIGURE 13.1  EVOLUTION OF LABORATORY ABNORMALITIES (ANC, PLATELETS, AST, ALT,……) ...... 43 
FIGURE 13.2  OVERALL SURVIVAL.......................................................................................................... 43 
FIGURE 13.3  PROGRESSION-FREE SURVIVAL. ......................................................................................... 44 
FIGURE 13.4  DURATION OF RESPONSE. .................................................................................................. 44 
FIGURE 13.5  WATERFALL PLOTS. .......................................................................................................... 45 
FIGURE 13.6  SPIDER PLOT (SLD) ........................................................................................................... 45 
FIGURE 13.7  BOXPLOT OF LVEF EVOLUTION FROM BL. ....................................................................... 46 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
10. 
Appendix I. Patient disposition and protocol deviations 
10.1. General characteristics 
10.1.1. Patients disposition 
Table 10.1.1.1  Patients disposition by institution. 
 
 
  Institution 1 
   Institution 2 
…. 
        Total 
   N        % 
    N 
   % 
    N 
  % 
    N 
   % 
N-eligible 
 
 
 
 
N-included 
N-treated 
      N-evaluable for safety 
         N-evaluable for efficacy 
NOTE: See listings from 10.1.1.2 to 10.1.1.5 
 
Table 10.1.1.2  Listing of patients not eligible. 
Patient ID 
Reason  
 
 
 
Table 10.1.1.3  Listing of patients included but not treated. 
Patient ID 
Reason  
 
 
 
Table 10.1.1.4  Listing of patients not evaluable for safety. 
Patient ID 
Reason  
 
 
 
Table 10.1.1.5  Listing of patients not evaluable for efficacy. 
Patient ID 
Reason  
 
 
 
10.1.2. Study dates 
Table 10.1.2.1  Study dates. 
Event 
        Date 
Date first consent 
 
Date first dose of first patient 
 
Date last consent 
 
Date first dose of last patient 
 
Date last dose  
 
Last follow up date 
 
10.1.3. Reasons for treatment discontinuation 


 
 
Table 10.1.3.1  Reasons for treatment discontinuation. 
 
Total 
N 
% 
   Progressive disease 
 
 
   Toxicity (Drug related AE)* 
 
 
   Death** 
 
 
   Patient refusal 
 
 
   Investigator decision 
 
 
   Other*** 
 
 
(*) See tables 10.1.3.6; (**) Table 10.1.3.7; (***) Table 10.1.3.5  
 
Table 10.1.3.2  Reasons for treatment discontinuation by number of cycles received. 
 
 
Cycle 1 
Cycle 2 
…. 
Total 
N 
% 
N 
% 
N 
% 
   N 
% 
   Progressive disease 
 
 
 
 
 
 
 
 
   Patient refusal 
 
 
 
 
 
 
 
 
       …. 
 
 
 
 
 
 
 
 
 
Table 10.1.3.3  Time on treatment (months). 
 
N  
Median* 
    Min*                          Max* 
 
 
 
 
(*) Time from first infusion of the study drug to the day of the last study drug administration 
plus 30 days, or onset of subsequent therapy or death, whichever occurs first. 
 
Table 10.1.3.4  Listings of patients and time on treatment. 
Patient ID. Day of first 
infusion 
Day of last dose 
administered 
Number of cycles 
received 
Time on treatment 
(months) 
 
 
 
 
 
 
Table 10.1.3.5  Listings of treatment discontinuation other than progressive disease. 
Patient ID. 
Cycle 
Reason 
Specify 
 
 
 
 
 
Table 10.1.3.6  Listing of treatment discontinuation due to adverse events (AEs). 
Patient ID. 
PT MedDRA Code 
Grade 
… 
Relationship 
Significant  
consequence 
 
 
 
 
 
 


 
 
 
Table 10.1.3.7  Listing of treatment discontinuation due to death. 
Patient ID.  Cycle 
Cause of 
death 
Last study drug 
administration  
date 
Death  
date 
Time from last 
Study drug administration  
to death (days) 
 
 
 
 
 
 
10.1.4. Reasons for patient’s study discontinuation 
Table 10.1.4.1  Reason for study discontinuation.  
 
Total 
N 
% 
Study termination 
 
 
Investigator decision 
 
 
Lost to follow up 
 
 
Death 
 
 
Never treated 
 
 
Patient refusal 
 
 
Other*  
 
 
 (*)See table 10.1.4.2 
 
Table 10.1.4.2  Listing of other reasons study discontinuation. 
Patient ID 
Reason 
Specify 
 
 
 
10.1.5. Protocol deviations 
Table 10.1.5.1  Eligibility: Patients who entered the study even though they did not satisfy the 
entry criteria. 
Patient ID 
Deviation type* 
Deviation description** 
 
 
 
(*) Including but not restricted to: Inclusion/Exclusion deviations, study procedures  
not performed, patients developing withdrawal criteria who were not withdrawn, patients who 
received incorrect dose or schedule, patients who received an excluded concomitant medication, 
and patients who do not satisfy criteria for treatment continuation. 
(**) Deviations with no effects on the risk/benefit ratio of the clinical trial will be distinguished 
from those that might have an effect on this risk/benefit ratio. 
 


 
 
11. 
Appendix II. Demographic analysis and efficacy evaluation 
11.1. Baseline characteristics 
11.1.1. Patients characteristics at baseline 
Table 11.1.1.1  Baseline characteristics: Summary statistics. 
 
Total (N=XX) 
N 
% 
Gender 
 
 
Male 
 
 
Female 
 
 
Age grouped: 
 
 
16 – XX years 
 
 
XX – YY years 
 
 
     ≥ YY years 
 
 
 
Table 11.1.1.2  Summary statistics: baseline characteristics. 
 
         N 
      Median 
Min 
 Max 
Age (years) 
 
 
 
 
11.1.2. Histology and time from diagnosis 
Table 11.1.2.1  Tumor histology. 
 
Total (N=XX) 
N 
% 
Tumor diagnosis:  
 
 
 Ewing’s bone sarcoma 
 
 
 Extraosseous Ewing: 
   PNET 
 
 
   Askin  
 
 
   Other* 
 
 
Primary tumor site:  
 
 
Upper extremity 
Lower extremity 
Face and neck 
 
 
Trunk/abdominal wall 
 
 
Other** 
 
 
Current Disease 
 
 
Unresectable locally advanced disease 
 
 
Metastatic 
 
 
Number of sites involved (target-non target):  
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
Summary statistics of number of sites involved (target-non 
target):  
 
 
N 
 
 
Median 
 
 
Minimum 
 
 


 
 
Maximum 
 
 
Programming Notes: Percentage is based on total patient included. 
(*)  and  (**) See listing 11.1.2.3 
 
Table 11.1.2.2  Sites for target and non-target lesions. 
Sites 
Total 
N 
% 
Bone 
 
 
Lung 
 
 
…. 
 
 
 
Table 11.1.2.3  Listing of patient histology. 
Patient ID. Tumor 
diagnosis 
Primary tumor  
site 
TNM at 
Diagnosis 
Stage at 
Diagnosis 
Other, 
Specify 
Current 
stage 
 
 
 
 
 
 
 
 
Table 11.1.2.4  Listing of target and non target lesions. 
Patient ID. 
Target/non-target 
Site/Subsite 
Method 
 
 
 
 
 
Table 11.1.2.5 Summary statistics of cancer diagnosis characteristics: Time from diagnosis and 
time from last progression disease (PD) to first infusion of study drug. 
 
N 
   Median 
        Min 
Max 
Time from first diagnosis to first infusion 
(months) 
 
 
 
 
 
 
 
 
Time from last progression to first infusion 
(months) 
 
 
 
 
 
 
 
 
11.1.3. Prior anticancer therapy 
Table 11.1.3.1  Surgery (palliative + curative). 
 
N 
% 
Yes  
 
 
No 
 
 
 
Table 11.1.3.2  Radiotherapy. 
 
N 
% 
Yes  
 
 
No 
 
 
 
Table 11.1.3.3  Patients with prior surgery. 
Patient ID. Intention 
Site and Procedures 
Surgery Date 
Residual disease 
 
 
 
 
 
 


 
 
Table 11.1.3.4  Summary statistics of prior systemic anticancer therapy. 
 
      N 
 Median 
       Min 
     Max 
Number of lines  
 
 
 
 
Number of agents 
 
 
 
 
 
Table 11.1.3.5  Number of lines and agents of systemic anticancer therapy. 
 
Total (N=XX) 
N 
% 
Number of lines 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
Number of agents 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
 
Table 11.1.3.6  Summary of lines and agents of systemic anticancer therapy. 
Antineoplastic and immunomodulating agents  
(ATC-clas. Levels 1-4) 
N 
% 
Antineoplastic Agents (L01) 
 
 
….. 
 
 
….. 
 
 
 
11.1.4. Prior history 
 
Table 11.1.4.1  Prior history. 
Patient ID. 
Description 
Onset date 
Resolved date 
 
 
 
 
 


 
 
11.1.5. Physical examination, vital signs, electrocardiogram and other tests 
 
Table 11.1.5.1  Physical examination at baseline. 
 
Total (N=XX) 
N 
% 
Physical Examination:  
 
 
Normal 
 
 
Abnormal 
 
 
ECOG-PS: 
 
 
0 
 
 
1 
 
 
2 
 
 
… 
 
 
ECG:  
 
 
Normal 
 
 
Abnormal 
 
 
LVEF 
 
 
Normal 
 
 
Abnormal 
 
 
(*) NA stands for “Not applicable” and specify reasons will be giving in a supportive listing. 
 
Table 11.1.5.2  Listings of patients with positive pregnancy test. 
Patient ID. 
Sample date 
 
 
 
Table 11.1.5.3  Listings of patients with no adequate contraception. 
Patient ID. 
Sample date 
 
 
 
Table 11.1.5.4   Listing of ECOG performance status. 
Patient ID. 
ECOG                
Specify* 
 
 
 
(*) If available details will be extracted from signs and symptoms. 
 
Table 11.1.5.5  Listing of patients with abnormal ECG at baseline. 
Patient ID. 
ECG result                
Specify* 
 
 
 
(*) If available details will be extracted from signs and symptoms. 
 
Table 11.1.5.6  Listing of patients with LVEF lower than the normal range. 
Patient ID. 
LVEF (%) 
Result 
Method   Institutional normal range 
 
 
 
 
 
Table 11.1.5.7  Summary statistics: Physical examination and vital signs. 


 
 
Parameter 
N 
Median 
Min 
  Max 
Weight (Kg) 
 
 
 
 
Height (cm) 
 
 
 
 
Heart rate (beats/min) 
 
 
 
 
BPS (mmHg) 
 
 
 
 
BPD (mmHg) 
 
 
 
 
Temperature (ºC) 
 
 
 
 
11.1.6. Hematological values at baseline 
Table 11.1.6.1  Hematological abnormalities at baseline. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
N              % 
Anemia 
 
 
 
 
 
 
 
 
 
Lymphopenia 
 
 
 
 
 
 
 
 
 
Neutropenia 
 
 
 
 
 
 
 
 
 
Thrombocytopenia 
 
 
 
 
 
 
 
 
 
(*) All hematological abnormalities susceptible to be graded in NCI-CTCAE v4.0  
 
Table 11.1.6.2  Hematology at baseline. Last value before treatment. 
Parameter* 
N 
Median 
Min 
Max 
Hemoglobin  
 
 
 
 
Lymphocytes 
 
 
 
 
Neutrophils  
 
 
 
 
Platelets  
 
 
 
 
(*)  Units for Leukocytes, Lymphocytes, Neutrophils and Platelets are in 109/l. Hemoglobin  
in g/dl 
 
Table 11.1.6.3  Hematology at baseline: Patients with hematological parameters missing. 
Patient ID. 
Parameter 
 
 
 
Table 11.1.6.4  Hematology at baseline. Abnormalities grade ≥ 2. 
Patient ID. 
Parameter 
Value      
Grade 
 
 
 
 
11.1.7. Biochemical values at baseline 
Table 11.1.7.1  Biochemical abnormalities at baseline. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
 N            % 
AST  
 
 
 
 
 
 
 
 
 
ALT 
 
 
 
 
 
 
 
 
 
*….. 
 
 
 
 
 
 
 
 
 
(*) Total bilirubin, AP, CPK and Creatinine. All biochemical abnormalities susceptible to be 
 graded in NCI-CTCAE v4.0  
 
Table 11.1.7.2  Biochemistry at baseline. Last value before treatment.  


 
 
Parameter* 
        N 
       Median 
Min 
Max 
AST 
 
 
 
 
ALT 
 
 
 
 
Total Bilirubin 
 
 
 
 
…. 
 
 
 
 
(*)  Units for AST, ALT, AP, CPK are IU/l. Units for Total Bilirubin and Creatinine are mg/dl.  
 
Table 11.1.7.3  Biochemistry at baseline. Patients with missing biochemical parameters. 
Patient ID. 
      Parameter 
 
 
 
Table 11.1.7.4  Biochemistry at baseline. Abnormalities grade ≥ 2. 
Patient ID. 
Parameter 
     Value      
Grade 
 
 
 
 
 
11.1.8. Other metabolic values at baseline 
Table 11.1.8.1  Other metabolic abnormalities at baseline (hyper-hypo). 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
 N            % 
Hyperglycemia 
 
 
 
 
 
 
 
 
 
*….. 
 
 
 
 
 
 
 
 
 
(*) Hyper and hypo abnormalities and all metabolic parameters susceptible to be graded  
as per NCI-CTCAE v4.0; i.e, Glucose, Sodium, Potassium, Calcium, Magnesium and Albumin. 
 
Table 11.1.8.2  Other metabolic abnormalities at baseline. Last value before treatment. 
Parameter* 
N 
Median 
Min 
Max 
Glucose 
 
 
 
 
 (*) Units for Glucose, Sodium, Clorhide, Potassium, Calcium, Magnesium are in mmol/l; 
Albumin and Total proteins in g/dl and LDH in IU/l; Creatinine Clearance (CrCl) in ml/min.   
 
Table 11.1.8.3  Patients with missing metabolic parameters. 
Patient ID. 
 Parameter 
 
 
 
Table 11.1.8.4  Other metabolic at baseline. Abnormalities grade ≥ 2. 
Patient ID. 
Parameter 
Value      
 Grade 
 
 
 
 
 
 
11.1.9. Signs and symptoms at baseline 
Table 11.1.9.1  Summary statistics: Signs and symptoms. 
SOC/MedDRA 
    Grade 1     Grade 2 
Grade 3 
Grade 4 
Total (N=XX) 


 
 
Preferred term 
     N 
 % 
N 
% 
N 
% 
N 
%     N            % 
General  
Disorders 
Abdomina
Distension
 
 
 
 
 
 
 
 
 
 
 
….. 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
Pain 
 
 
 
 
 
 
 
 
 
 
 
 
Table 11.1.9.2  Median of signs and symptoms  
 
N 
Median 
Min 
Max 
 
 
 
 
 
 
Table 11.1.9.3  Listing of baseline characteristics: Signs and symptoms (Grade ≥ 2). 
Patient 
ID 
SOC 
Sign/symptom 
(PT) 
Verbatim   
term 
Grade 
Onset 
date 
Relationship 
Treated 
 
 
 
 
 
 
 
 
 
11.1.10. Concomitant therapy 
Table 11.1.10.1  Agents of concomitant therapy started at baseline. 
Concomitant medication (ATC1/ATC2/ATC4) 
 
System(ATC1) 
  Indication(ATC2) 
   Agents(ATC4) 
N  
% 
Alimentary tract 
and metabolism 
 
 
 
Stomatological 
preparations 
 
 
Antiinfectives for local 
oral treat. 
Other agents for …. 
………… 
 
 
Antiacids 
 
………. 
……… 
 
 
 
 
Table 11.1.10.2  Concomitant medication characteristics started at baseline. 
 
Total (N=XX) 
N 
% 
No of systems (ATC1) 
 
 
   0 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
No of indications (ATC2) 
 
 
   0 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
No of agents (ATC4) 
 
 
   0 
 
 
   1 
 
 
   2 
 
 


 
 
   3 
 
 
> 3 
 
 
 
Table 11.1.10.3  Concomitant therapy started at baseline. Summary of number, indications and 
systems involved. 
Concomitant medication 
(ATC1/ATC2/ATC4)     
N 
Median 
Min 
Max 
Number of systems involved (ATC1) 
 
 
 
 
Number of indications involved (ATC2) 
 
 
 
 
Number of agents involved (ATC4) 
 
 
 
 
 
11.2. Efficacy 
11.2.1. Response 
Table 11.2.1 1  Patients evaluable for efficacy. 
Evaluable 
Total (N=XX) 
N 
% 
Yes 
 
 
No 
 
 
Total 
 
 
 
Table 11.2.1.2  Listing of patients excluded from the efficacy analysis. 
Patient ID 
Reason 
 
 
 
Table 11.2.1.3  Overall response rate. 
Response 
N 
%              
First stage 
 
 
           CR+PR 
 
 
Total 
 
 
           CR+PR 
 
 
 
Table 11.2.1.4  Best response in all evaluable patients. 
Response 
             N 
         % 
Complete Response (CR) 
 
 
Partial Response (PR) 
 
 
Stable Disease (SD) 
 
 
Progression Disease (PD)* 
 
 
Treatment Failure 
 
 
Programming Notes: Percentage is based on the total of patient evaluable for efficacy. 
 (*)  Patients with early PD will also be included in this category (provided that they receive 
 at least two infusions of Zalypsis®) 
 


 
 
Table 11.2.1.5  Best response in all treated patients. 
Response 
N 
             % 
Complete Response (CR) 
 
 
Partial Response (PR) 
 
 
Stable Disease (SD) 
 
 
Progression Disease (PD)* 
 
 
Treatment failure 
 
 
    Not Evaluable (NE) 
 
 
Programming Notes: Percentage is based on total patient of patients treated 
(*)  Patients with early PD will also be included in this category (provided that they receive 
 at least two infusions of Zalypsis®) 
 
Table 11.2.1.6  Characteristics of responders  
Patient characteristics* 
Outcome prior treatment** 
PM00104 treatment*** 
 
 
 
(*)Patient ID, Sex/Age/PS and Histology. (**)No. prior CT lines, last prior treatment, cycles 
received and best response. *** TTP, Cycles with PM104, PFS, OS. 
11.2.2. Time-to-event variables  
Table 11.2.2.1  Duration of response. 
Summary* 
N=XX 
Events X (XX.X%) 
Censored X (XX.X%) 
Median XX 95% CI (XX.X-X.X) 
(*) Responder patients by RECIST v1.1, i.e, CR and PR. 
 
Table 11.2.2.2  Progression-free survival (Kaplan-Meier estimates in all evaluable patients). 
Summary 
N=XX 
Events X (XX.X%) 
Censored X (XX.X%) 
Median XX 95% CI (X.X-X.X) 
PFS rate at 3 months XX.X% 95% CI (XX.X%-XX.X%) 
PFS rate at 6 months XX.X% 95% CI (XX.X%-XX.X%) 
 
Table 11.2.2.3  Overall survival. (Kaplan-Meier estimates in all evaluable patients). 
Summary 
N=XX 
Events X (XX.X%) 
Censored X (XX.X%) 
Median X.X 95% CI (XX.X-X.X) 
OS rate at 12 months XX.X% 95% CI (XX.X%-XX.X%) 
 
Table 11.2.2.4  Progression-free survival (Kaplan-Meier estimates in all treated patients). 


 
 
Summary 
N=XX 
Events X (XX.X%) 
Censored X (XX.X%) 
Median XX 95% CI (X.X-X.X) 
PFS rate at 3 months XX.X% 95% CI (XX.X%-XX.X%) 
PFS rate at 6 months XX.X% 95% CI (XX.X%-XX.X%) 
 
 
Table 11.2.2.5  Overall survival. (Kaplan-Meier estimates in all treated patients). 
Summary 
N=XX 
Events X (XX.X%) 
Censored X (XX.X%) 
Median X.X 95% CI (XX.X-X.X) 
OS rate at 12 months XX.X% 95% CI (XX.X%-XX.X%) 
 
 
Table 11.2.2.6  Follow-up period. 
  Parameter 
 Median 
   95% CI Lower bound 
95% CI Upper bound 
    PFS 
 
 
 
    OS 
 
 
 
 
12. 
Appendix III. Safety evaluation 
12.1. Extent of exposure 
12.1.1. Treatment administration  
Table 12.1.1.1  Cycles administered. 
Cycles per patient: n (%) 
 
N 
% 
1 
 
 
2 
 
 
3 
 
 
… 
 
 
 
Table 12.1.1.2  Summary of cycles administered. 
 
N 
Median  
Min 
Max  
 
 
 
 
 
 
Table 12.1.1.3  Dose intensity. 
    
N 
Median 
Min 
Max 
Cumulative dose (mg/m²) 
 
 
 
 
Dose intensity (mg/m² wk) 
 
 
 
 
Relative dose intensity (%) 
 
 
 
 


 
 
 
12.1.2. Dose administration delays 
 
Note: Dose delays refer to delays in the administration of the first infusion of one cycle (Day 
1 infusion).  
Table 12.1.2.1  Dose delay 
 
N 
% 
Number of patients treated 
 
 
Number of patients susceptible of delay* 
 
 
Number of cycles administered 
 
 
Number of cycles susceptible of delay* 
 
 
Patients** with 
 
 
No delays 
 
 
1 cycle delayed 
 
 
2 cycles delayed 
 
 
 3 cycles delayed 
 
 
Number of cycles with dose delay***  
 
 
Programming Notes: Percentage is based on number of cycle susceptible to be delayed. 
(*) Patients who receive only one cycle of Zalypsis®. 
(**)  Denominator: Number of patients susceptible of delay = (Total number of patients* – 
 Number of patients with first cycle only) 
(***) Denominator: Number of cycles susceptible of delay = (Number of cycles administrated 
 – Total number of first cycles)  
 
Table 12.1.2.2  Dose delay according to their relation to study drug. 
 
Treatment related*** 
Non – Treatment related 
N 
% 
N 
% 
Patients* with 
 
 
 
 
No delays 
 
 
 
 
1 cycle delayed 
 
 
 
 
2 cycles delayed 
 
 
 
 
 3 cycles delayed 
 
 
 
 
Number of cycles with dosing delay *  
 
 
 
 
Programming Notes: 
(*) Patients who receive only one infusion of Zalypsis® and have treatment discontinuation  
within the first cycle due to any cause except for drug related AE are not susceptible of delay. 
(**) Denominator = Number of cycles susceptible to be delayed. 
(***) Hematological reason / Non-hematological reason. 
 
Table 12.1.2.3  Length of dose delay. 
 
Treatment related** 
  Non – Treatment related 
   Total 
      N 
       % 
         N 
       % 
 N 
% 
Length of delay* 
 
 
 
 
 
 
No delay 
 
 
 
 
 
 
≤ 7 days 
 
 
 
 
 
 
8 – 14 days 
 
 
 
 
 
 
 14 days 
 
 
 
 
 
 


 
 
Programming Notes: 
(*) Denominator = Number of cycles susceptible to be delayed. 
(**) Hematological reason / Non-hematological reason. 
 
Table 12.1.2.4  Dose delay and reason by cycle. 
 
Cycle 2 
Cycle 3 
… 
Total 
 N 
% 
N 
% 
N 
% 
N 
% 
Number of delays 
 
 
 
 
 
 
 
 
No delay 
 
 
 
 
 
 
 
 
Treatment related* 
 
 
 
 
 
 
 
 
 Hematological reason  
 
 
 
 
 
 
 
 
 Non-hematological reason
 
 
 
 
 
 
 
 
Non-treatment related* 
 
 
 
 
 
 
 
 
Programming Notes: (*) Denominator = Number of cycles susceptible to be delayed. 
 
Table 12.1.2.5  Listing of patient with dose delay. 
Patient 
ID. 
Reason 
for 
dose 
delay 
Dose 
Delay 
Spec 
Delayed 
Cycle 
Delayed 
cycle 
start 
date 
Previous 
cycle 
Previous 
cycle 
start 
date 
Dose Delay 
calculated. 
(days) 
 
 
 
 
 
 
 
 
 
Table 12.1.2.6  Listing of patients with dose interruption and/or re-administration 
Patient ID. 
Cycle 
Reason 
Date 
Intended dose 
Total dose 
Total volume 
 
 
 
 
 
 
 
 
12.1.3. Skipped doses 
Note: Infusions not administered on Days 8 and/or 15 of the cycle are considered skipped 
doses. 
Table 12.1.3.1  Skipped doses. 
 
N 
% 
Number of patients treated with at least 2 infusions 
 
 
Number of patients susceptible to have a omission 
 
 
Number of patients with any skipped dose* 
 
 
Number of patients with any skipped dose (drug related) 
 
 
Number of cycles administered 
 
 
Number of cycles with at least one skipped dose (drug 
related) 
 
 
Day 8 
 
 
Day 15 
 
 
     Day 8 +15 
Number of cycles with  
at least one skipped dose 
Treatment related** 
Non–treatment related 
N 
% (of all cycles) 
N 
% (of all cycles) 


 
 
Day 8 
 
 
 
 
Day 15 
 
 
 
 
Day 8 +15  
 
 
 
 
 (*) Patients who receive only one infusion of Zalypsis® and have treatment discontinuation 
within the first cycle due to any cause except for drug related AE are not susceptible of dose 
omission 
 (**)  Hematological reason / Non-hematological reason. 
 
 
Table 12.1.3.2  Listing of patients with skipped doses. 
Patient ID. Cycle/infusion  
(skipped dose)  
 Reason (drug related/other) 
Comments 
 
 
 
 
 
 
12.1.4. Dose reduction 
Note: All dose reductions will be considered and described, specifying the reason for 
reduction (hematological toxicity, non-hematological toxicity or other causes 
          not related to study drug). 
Table 12.1.4.1  Dose reduction. 
 
N 
% 
Number of patients treated 
 
 
Number of patients susceptible of reduction* 
 
 
Number of cycles administered 
 
 
Number of cycles susceptible of reduction* 
 
 
Patients ** with 
 
 
No reductions 
 
 
1 cycle reduction 
 
 
2 cycle reductions 
 
 
 3 cycle reductions 
 
 
Number of cycles with dose reduction *** 
 
 
Programming Notes: Percentage is based on number of cycle susceptible to be reduced. 
(*) Patients who receive only one infusion of Zalypsis® and have treatment discontinuation  
within the first cycle due to any cause except for drug related AE are not susceptible of delay. 
(**)   Denominator = Number of patients susceptible of reduction = (Total number of patients 
 – Number of patients with first cycle only) 
(***) Denominator = Number of cycles susceptible of reduction = (Number of cycles  
administered – Total number of first cycles) 
 
Table 12.1.4.2  Dose reduction according to their relation to study drug. 
 
Treatment related** 
Non – Treatment related 
N 
% 
N 
% 
Patients* with 
 
 
 
 
  No dose reductions 
 
 
 
 
  1 dose reduction 
 
 
 
 
  2 dose reductions 
 
 
 
 
   3 dose reductions 
 
 
 
 


 
 
Number of cycles with dose 
reduction*  
 
 
 
 
Programming Notes: (*) Denominator = Number of patients/cycles susceptible to have had a  
dose reduced. (**) Hematological reason/Non-hematological reason. 
 
Table 12.1.4.3  Dose reduction and reason by cycle. 
 
     Cycle 2 
      Cycle 3 
… 
     Total 
N 
% 
N 
% 
N 
% 
N 
% 
Number of patients 
 
 
 
 
 
 
 
 
Number of reductions 
 
 
 
 
 
 
 
 
No reductions 
 
 
 
 
 
 
 
 
Treatment-related* 
 
 
 
 
 
 
 
 
    Hematological reason  
 
 
 
 
 
 
 
 
    Non-hematological reaso
 
 
 
 
 
 
 
 
Non-Treatment related* 
 
 
 
 
 
 
 
 
Programming Notes. (*) Denominator = Number of patients susceptible to have had a  
dose reduced (who have received more than one cycle). 
 
Table 12.1.4.4  Patients with dose reduction. 
Patient 
ID 
Reason for  
dose  
reduction 
Dose  
reduction  
Spec 
Dose in 
Reduction 
cycle 
Dose in 
Previous 
cycle 
Dose reduction  
Calculated (%) 
 
 
 
 
 
 
 
Table 12.1.4.5  Patients with dose delay and reduction. 
Patient ID. Cycle  
Reason  
 Comments 
Dose delay (days) 
(%) Reduction  
 
 
 
 
 
 
 
12.2. Safety  
12.2.1. Adverse events 
Table 12.2.1.1  Summary of adverse events. 
Number of patients* with 
N 
% 
Any AE  
 
 
Any drug-related* AE  
 
 
Any AE grade ≥ 3 
 
 
Any drug-related AE grade ≥ 3 
 
 
Any SAE 
 
 
Any drug-related SAE 
 
 
Deaths associated with AEs  
 
 
Deaths associated with drug-related AEs  
 
 
Discontinuations associated with AEs  
 
 
Discontinuations associated with drug-related AEs 
 
 
Dose delays associated with AEs 
 
 
Dose delays associated with drug-related AEs 
 
 
Dose skipped associated with AEs 
 
 


 
 
Dose skipped associated with drug-related AEs 
 
 
Dose reduction associated with AEs 
 
 
Dose reduction associated with drug-related AEs 
 
 
 (*) Safety Population ; (**) Drug related and unknown  
 
Table 12.2.1.2  Summary of hematological laboratory abnormalities. 
Number of patients* with** 
N 
% 
Any hematological abnormality  
 
 
Any hematological abnormality grade ≥ 3 
 
 
 (*) Safety Population.(**) Includes only Hemoglobin, absolute neutrophil count (ANC)  
and Platelets.  
 
Table 12.2.1.3  Summary of non-hematological laboratory abnormalities. 
Number of patients* with** 
N 
% 
Any non-hematological abnormality  
 
 
Any hematological abnormality grade ≥ 3 
 
 
 (*) Safety Population. (**) Includes only ALT, AST, AP, Total Bilirubin, CPK, Creatinine.  
 
Table 12.2.1.4  Drug-related adverse events. Worst case per patient 
Table 12.2.1.5  Drug-related adverse events occurring in > 10% Worst case per patient. 
Table 12.2.1.6  All adverse events regardless relationship. Worst case per patient. 
Table 12.2.1.7  All adverse events regardless relationship occurring in > 10% Worst case per 
patient. 
Table 12.2.1.8  Drug-related adverse events. Worst case per cycle. 
Table 12.2.1.9  Drug-related adverse events occurring in > 10% Worst case per cycle. 
Table 12.2.1.10  All adverse events regardless relationship. Worst case per cycle. 
Table 12.2.1.11  All adverse events regardless relationship occurring in > 10% Worst case per 
cycle. 
Table 12.2.1.12  Listing of AEs leading to death. 
Table 12.2.1.13  Listing of AEs treatment related leading to death. 
Table 12.2.1.14  Listing of AEs leading to dose delay. 
Table 12.2.1.15  Listing of AEs leading to dose reduction. 
Table 12.2.1.16  Listing of AEs leading to dose delay and reduction. 
 
Tables from 12.2.1.4 to 12.2.1.11 will have the following format and grades could be grouped 
into categories if required. 
System Organ Class/ 
Preferred Term 
Grade 1 
Grade 2 
Grade 3 Grade 4 
   Total (G1-G4) 
 
N 
% N 
% N 
% N 
%   N 
 % 
System Organ Class 1 (Total) 
 
 
 
 
 
 
 
 
 
 
             Preferred Term 1 
 
 
 
 
 
 
 
 
 
 
System Organ Class 2 (Total) 
 
 
 
 
 
 
 
 
 
 


 
 
             Preferred Term 1 
 
 
 
 
 
 
 
 
 
 
 …….. 
 
 
 
 
 
 
 
 
 
 
Percentages are based on the safety population. Table shows number of distinct patients with 
events. 
 
Tables from 12.2.1.12 to 12.2.1.16 have the following format*: 
Patient ID.  
Verbatim 
name 
MedDRA 
Preferred 
term 
Cycle 
Grade 
……. 
Onset 
date 
 End 
Date   ........ 
 
 
 
 
 
 
 
 
 (*) Delays and reductions will be checked with information in drug administration.  
 
12.2.2. Grade 3-4 adverse events 
Table 12.2.2.1  Listing of patients with grade 3-4 AEs (treatment-related). 
Patient 
ID. 
Cycle 
MedDRA 
preferred 
term 
Verbatim 
name 
Grade 
Relationship 
Onset 
date 
…. 
 
 
 
 
 
 
 
 
(*)End date, Action taken and serious criteria 
 
Table 12.2.2.2  Listing of patients with grade 3-4 AEs (treatment-related). 
Patient 
ID. 
Cycle 
MedDRA 
preferred 
term 
Verbatim 
name 
Grade 
Relationship 
Onset 
date 
…. 
 
 
 
 
 
 
 
 
(*)End date, Action taken and serious criteria 
12.2.3. Deaths and serious adverse events (SAEs) 
 
All serious adverse events will be listed only for the purpose of reconciliation with the database 
of the Pharmacovigilance department. The listings provided by the Pharmacovigilance 
department will be used for the clinical study report. 
Table 12.2.3.1  Listing of SAEs. 
Patient 
ID. 
Cycle 
MedDRA 
preferred 
term 
Verbatim 
name 
Grade 
Relationship 
Onset 
date 
End 
date 
 
 
 
 
 
 
 
 
 (*) Also Action taken and serious criteria 
 
Table 12.2.3.2  Listing of all deaths. 
Patient 
ID. 
Cycle 
MedDRA 
preferred 
term 
Verbatim 
name 
Grade 
Relationship 
Onset 
date 
End 
date 
 
 
 
 
 
 
 
 


 
 
 (*)Taken from death report form.(**)Also Time on treatment: defined as last infusion date plus 
30 days, or date of death or subsequent therapy (whichever comes first) minus first infusion date 
and Time from last dose defined as death date minus last infusion date. 
 
12.3. Laboratory evaluation 
12.3.1. Hematological abnormalities  
Table 12.3.1.1  Hematological abnormalities during treatment, worst grade per patient. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
     N         % 
Anemia 
 
 
 
 
 
 
 
 
 
Lymphopenia 
 
 
 
 
 
 
 
 
 
Neutropenia 
 
 
 
 
 
 
 
 
 
Thrombocytopenia 
 
 
 
 
 
 
 
 
 
Programming Notes: Percentages are based on the safety population. (*) All hematological 
abnormalities susceptible to be graded in NCI-CTCAE v4.0. 
 
Table 12.3.1.2  Hematological abnormalities during treatment, worst grade per cycle. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
    N            % 
Anemia 
 
 
 
 
 
 
 
 
 
Lymphopenia 
 
 
 
 
 
 
 
 
 
Neutropenia 
 
 
 
 
 
 
 
 
 
Thrombocytopenia 
 
 
 
 
 
 
 
 
 
Programming Notes: Percentages are based on the safety population. 
 (*) All hematological abnormalities susceptible to be graded in NCI-CTCAE v4.0. 
 
Table 12.3.1.3  Patients with any hematological abnormalities grade 3-4. 
Patient 
    ID 
Cycle Event/ 
Parameter*
Value 
at BL* 
Grade 
 at BL 
Onset 
day 
Onset 
value 
Onset 
value 
…** 
Days with  
Grade 3-4 
 
 
 
 
 
 
 
 
 
 
(*) Units for Leukocytes, Lymphocytes, Neutrophils and Platelets are in 109/l units in %  
and Hemoglobin in g/dl. (**) Also Nadir day, nadir value, recovery date, recovery  
value, recovery grade. 
 
Table 12.3.1.4  Listing of patients and cycles with hematological evaluations missing. 
Patient ID 
       Cycle 
         Parameter  
 
 
 
 


 
 
 
Table 12.3.1.5  Time course and recovery from hematological toxicities. 
 
 
N 
All cycles 
 
Median 
Range 
Neutrophils 
 
 
 
Day of nadir neutrophils count  
 
 
 
Nadir of neutrophils count (/mm3) 
 
 
 
Day of neutropenia G3-4 onset 
 
 
 
Day of recovery to grade 2* 
 
 
 
Number of days with grade 3-4 neutropenia 
 
 
 
Platelets 
 
 
 
Day of nadir platelets count  
 
 
 
Nadir of platelets count (/mm3) 
 
 
 
Day of thrombocytopenia G3-4 onset 
 
 
 
Day of recovery to grade 2* 
 
 
 
Number of days with grade 3-4 thrombocytopenia   
 
 
 
Programming notes: (*) Denominator = No of cycles with Grade 3-4. Also recovery to  
grade 1 or an specific value if required. 
 
 
Table 12.3.1.6  Baseline grade and evolution of hematological toxicities. 
 
    Worst grade per patient during treatment 
Baseline 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
     Grade 4 
N 
% 
N 
% 
N 
% 
N 
% 
N 
% 
Anemia 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
 
 
 
 
 
 
 
 
 
Neutropenia 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
 
 
 
 
 
 
 
 
 
……. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12.3.2. Biochemical abnormalities  
 
Table 12.3.2.1  Biochemical abnormalities during treatment, worst grade per patient. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
   N            % 
AST  
 
 
 
 
 
 
 
 
 
ALT 
 
 
 
 
 
 
 
 
 
*….. 
 
 
 
 
 
 
 
 
 


 
 
Programming Notes: Percentages are based on the safety population. (*) All biochemical 
abnormalities susceptible to be graded by NCI-CTCAE v4.0.  
 
Table 12.3.2.2  Biochemical abnormalities during treatment, worst grade per cycle. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
% 
   N            % 
AST  
 
 
 
 
 
 
 
 
 
ALT 
 
 
 
 
 
 
 
 
 
*….. 
 
 
 
 
 
 
 
 
 
Programming Notes: Percentages are based on the safety population. (*) All biochemical 
abnormalities susceptible to be graded by NCI-CTCAE v4.0.  
 
Table 12.3.2.3  Supportive listing: Patients with any biochemical abnormalities grade 3-4. 
Patient 
ID 
Cycle 
Event/ 
Parameter 
 Value 
 at BL 
Grade 
 at BL 
Onset 
day 
Onset 
value 
…..* 
Days with  
Grade 3-4 
 
 
 
 
 
 
 
 
 
(*) Nadir day, nadir value, recovery date, recovery value and recovery grade to grade ≤ 2. 
 
Table 12.3.2.4  Listing of patients and cycles with biochemical evaluations missing. 
Patient ID 
      Cycle 
      Lab test  
 
 
 
 
Table 12.3.2.5  Time course and recovery from biochemical toxicities. 
 
 
N 
              All cycles 
 
Median 
Range 
ALT 
 
 
 
Day of peak ALT count  
 
 
 
Peak of ALT count (/mm3) 
 
 
 
Day of ALT G3-4 onset 
 
 
 
Day of recovery to grade 2 
 
 
 
Number of days with grade 3-4 ALT increase 
 
 
 
AST 
 
 
 
Day of peak AST count  
 
 
 
Peak of AST count (/mm3) 
 
 
 
Day of AST G3-4 onset 
 
 
 
Day of recovery to grade 2* 
 
 
 
Number of days with grade 3-4 AST increase 
 
 
 
Programming notes: (*) Denominator= Number of cycles with Grade 3-4. Also recovery to  
grade 1 if required. 


 
 
 
Table 12.3.2.6  Baseline grade and evolution of biochemical toxicities. 
 
    Worst grade per patient during treatment 
Baseline 
Grade 0 
Grade 1 
Grade 2 
     Grade 3 
      Grade 4 
 N
% 
N 
% 
N 
% 
N 
% 
N 
% 
AST 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
 
 
 
 
 
 
 
 
 
ALT 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
 
 
 
 
 
 
 
 
 
……. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12.3.3. Other metabolic abnormalities 
Table 12.3.3.1  Metabolic abnormalities during treatment, worst grade per patient 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
%    N 
   % 
Hyperglycemia 
 
 
 
 
 
 
 
 
 
 
…. 
 
 
 
 
 
 
 
 
 
 
(*) All metabolic parameters susceptible to be graded as per NCI-CTCAE v4.0  
 
Table 12.3.3.2  Metabolic abnormalities during treatment, worst grade per cycle.  
Parameter* 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
%    N 
   % 
Hyperglycemia 
 
 
 
 
 
 
 
 
 
 
…. 
 
 
 
 
 
 
 
 
 
 
(*) All metabolic parameters susceptible to be graded as per NCI-CTCAE v4.0  
 
Table 12.3.3.3  Metabolic abnormalities grade per cycle. 
Parameter* 
Grade 1 
Grade 2 
Grade 3 
    Grade 4 
Total (G1-G4) 
N 
% 
N 
% 
N 
% 
N 
%    N 
    % 
Hyperglycemia 
 
 
 
 
 
 
 
 
 
 
…. 
 
 
 
 
 
 
 
 
 
 
 (*) All metabolic parameters susceptible to be graded as per NCI-CTCAE v4.0  
 
Table 12.3.3.4  Listing of patients and cycles with metabolic evaluations missing. 
Patient ID. 
Cycle 
Lab. test 
 
 
 
 


 
 
12.4. Vital Signs 
Table 12.4.1 Vital signs. 
Vital Signs 
Baseline 
Cycle 1 
Cycle 2 
… 
Cycle (X) 
PS 
 
 
 
 
 
BPS (mmHg) 
 
 
 
 
 
BPD (mmHg) 
 
 
 
 
 
Weigth (Kg) 
 
 
 
 
 
 
12.5. Prophylactic and concomitant medication during treatment 
Table 12.5.1  Patients who received prophylactic medication. 
No patients 
treated 
No patients with  
Adequate 
prophylaxis 
% 
N Cycles  
administered 
No Cycles with 
adequate 
prophylaxis 
% 
 
 
Table 12.5.2  Prophylactic therapy by patient. 
Patient ID Cycles 
Dexamethasone 
Y/N 
Dose 
Ondansetron 
Y/N 
Dose 
Other   Specify 
 
Cycle 1 
 
 
 
 
 
 
 
Cycle 2 
 
 
 
 
 
 
 
Cycle n 
 
 
 
 
 
 
 
Table 12.5.3  Agents of concomitant therapy started during treatment. 
Concomitant medication (ATC1/ATC2/ATC4) 
 
 
System(ATC1) 
  Indication(ATC2) 
   Agents(ATC4) 
N  
% 
Alimentary tract 
and metabolism 
 
 
 
Stomatological 
preparations 
 
 
Antiinfectives for 
local oral treat. 
Other agents for …. 
………… 
 
 
Antiacids 
 
………. 
……… 
 
 
 
 
Table 12.5.4  Concomitant medication characteristics during treatment. 
 
Total (N=XX) 
N 
% 
Number of Systems (ATC1) 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
Number of indications (ATC2) 
 
 
   1 
 
 
   2 
 
 
   3 
 
 


 
 
> 3 
 
 
Number of agents (ATC4) 
 
 
   1 
 
 
   2 
 
 
   3 
 
 
> 3 
 
 
Programming Notes: Percentage is based on total patient included. 
 
Table 12.5.5  Summary of concomitant medication during treatment. 
Concomitant medication 
(ATC1/ATC2/ATC4)     
N 
Median 
Min 
Max 
Number of systems involved (ATC1) 
 
 
 
 
Number of indications involved (ATC2) 
 
 
 
 
Number of agents involved (ATC4) 
 
 
 
 
 
12.6. Cardiac safety 
Table 12.6.1  Listing of patients with Cardiac AEs and characteristics. 
Patient 
ID. 
Cycle* 
MedDRA 
PT 
Grade 
** 
Relationship 
….*** 
 
 
 
 
 
 
*Cycle in witch the AE occurred. **According to NCI-CTCAE v.4.0 *** Also Seriousness 
Criteria, Action taken… 
Table 12.6.2  Listing of ECG evolution per patient.  
Patient ID 
Baseline 
Cycle 1 
Cycle 2 
….. 
Cycle (X) 
 
 
 
 
 
 
 
Table 12.6.3  Listing of patients with abnormal ECG.  
Patient ID 
Result at baseline 
Cycle 
Specify* 
 
 
 
 
(*) If applicable information obtained from AE. 
 
Table 12.6.4  Median change in LVEF (%). 
 
        N 
       Median 
Min 
Max 
 
 
 
 
 
 
Table 12.6.5  Listing of LVEF/ECG evolution per patient.  
Patient ID. 
Value at 
baseline 
Cycle 1* 
Cycle 2* 
….. 
Cycle (X)* 
 
 
 
 
 
 
* ECG for day 1, day 8 and day 15. 
  
 


 
 
Table 12.6.6  LVEF characteristics per patient.  
Patient ID. 
Value at baseline 
Minimum value 
Cycle (minimum value) 
 
 
 
 
 
Table 12.6.7  Median change in Troponin I. 
 
    N 
       Median 
Min 
Max 
Baseline to nadir value (%) 
 
 
 
 
Baseline to last value (%) 
 
 
 
 
(*) Also tables in (x ULN) values if applicable. 
 
Table 12.6.8  Listing of Troponin I evolution. 
Patient ID. 
Value* at Baseline 
(ng/ml) 
Cycle 1 
Cycle 2 
….. 
Cycle (X) 
 
 
 
 
 
 
(*) Also tables in (x ULN) values if applicable 
 
Table 12.6.9  Troponin I characteristics per patient. 
Patient ID. 
Value* at baseline 
(ng/ml) 
Maximum value 
 Cycle (maximum value) 
 
 
 
 
(*) Also tables in (x ULN) values if applicable 
 
12.7. Other tests and procedures  
Table 12.7.1.1 Other tests and procedures. 
Patient ID. 
Cycle 
Test  Date 
Test name Result(Normal/Abnormal) Findings 
 
 
 
 
 
 


 
 
 
13. 
Appendix IV. Figures 
 
NOTE:  Figures here showed are examples and may vary in the format and will be showed for 
“all evaluable patients” and “all treated patients” when applicable. 
 
Figure 13.1  Evolution of laboratory abnormalities (ANC, Platelets, AST, ALT,……) 
 
 
 
 
Figure 13.2  Overall survival. 
 
 


 
 
 
Figure 13.3  Progression-free survival. 
 
Figure 13.4  Duration of response. 
 
Kaplan-Meier curve of duration of response (if any response is observed). 
 


 
 
 
Figure 13.5  Waterfall plots. 
 
 
Maximum tumor shrinkage in measurable lesions by RECIST v1.1 will be displayed. 
Additional graphs by histology or RECIST response will also be provided when applicable. 
 
Figure 13.6  Spider plot (SLD) 
 
 
(*) Sum of longest diameters for all target lesions.  


 
 
 
Figure 13.7  Boxplot of LVEF evolution from BL. 
 
Boxplot showing the change in LVEF from baseline, to worst value by cycle. 
 


 
 
14. 
Appendix V. DB listings. 
-Listing 14.1.1.  Cover 
-Listing 14.1.2.  Study registration 
-Listing 14.1.3.  Demography 
-Listing 14.1.4.  Pregnancy test 
-Listing 14.1.5.  Prior history 
-Listing 14.1.6.  Ewing sarcoma history 
-Listing 14.1.7.  Prior anticancer therapy: surgery  
-Listing 14.1.8.  Prior anticancer therapy: radiotherapy 
-Listing 14.1.9.  Prior anticancer systemic therapy: chemotherapy/biological therapy 
-Listing 14.1.10.  Drug administration 
-Listing 14.1.11.  Hematology laboratory values 
-Listing 14.1.12.  Biochemical laboratory values 
-Listing 14.1.13.  Other metabolic laboratory values 
-Listing 14.1.14.  Physical examination 
-Listing 14.1.15.  Performance status 
-Listing 14.1.16.  Vital signs 
-Listing 14.1.17.  Electrocardiogram 
-Listing 14.1.18.  LVEF 
-Listing 14.1.19.  Troponin I 
-Listing 14.1.20.  Other tests and procedures 
-Listing 14.1.21.  Adverse events (including signs and symptoms) 
-Listing 14.1.22.  Concomitant therapy/procedures 
-Listing 14.1.23.  Antiemetic prophylactic medication 
-Listing 14.1.24.  Tumor evaluation 
-Listing 14.1.25.  Radiological evaluation response assessment by cycle 
-Listing 14.1.26.  Clinical evaluation response assessment by cycle 
-Listing 14.1.27.  Overall response assessment by cycle 
-Listing 14.1.28.  Best overall response 
-Listing 14.1.29.  End of treatment 
-Listing 14.1.30.  Follow up 
-Listing 14.1.31.  Death report form 
-Listing 14.1.32.  Off study 
-Listing 14.1.33.  Signature report 
 
 


 
 
15. 
SAP Version History.  
 
Due to safety reasons, an independent cardiologist will undertake a review of all the ECGs and 
echocardiograms performed to the patients during the course of the study, in order to evaluate 
the cardiotoxicity risk of PM00104.  This analysis was originally included in the first version of 
the SAP but due to logistical reasons, this analysis will be performed lately the database 
analyses, consequently it will be done in a separate document. The Statistical Analysis 
Plan is changed in accordance to the new version (version 2) as follows:  
  
        All graphs and listings referred in the first version of the statistical plan will be 
presented in a separate document and will include data from CRF database and 
independent review database. 
 
 
In addition some minor changes and corrections in the text and to the mock shells have 
been done in order to improve a clear, unambiguous communication of the science and 
statistics of the trial. 
 
Major changes: 
 
Section 8.7 Independent review committee: 
 
Original text: “Because of safety reasons, an independent cardiologist will undertake a 
review of all the ECGs and echocardiograms performed to the patients during the course of the 
study, in order to evaluate the cardiac risk of Zalypsis®. For this purpose all tables, graphs and 
listings will be presented in duplicate and all results will specify from which database are 
obtained, that is, CRF database and independent review database.  
See section 12.6 Cardiac Safety.” 
 
Change to: “Because of safety reasons, an independent cardiologist will undertake a review of 
all the ECGs and echocardiograms performed to the patients during the course of the study, in 
order to evaluate the cardiotoxicity risk of PM00104. For this purpose, an independent 
cardiologist´s report not included in this SAP will be presented as an Appendix of the CSR”  
 
Section 12.6 Cardiac Safety: 
 
Original text: “As an independent cardiologist will undertake a review of all the ECGs and 
echocardiograms performed to the patients during the course of the study, tables and listings 
defined in this section will be duplicated and will have a comprehensive header indication 
which date base were obtained, i.e, CRF database and Independent review database. 
In this section additional tables and listings will be provided under request if clinical interest.” 
Change to: “As an independent cardiologist will undertake a review of all the ECGs, 
echocardiograms and troponin I determinations performed to the patients during the course of the 
study, tables and listings defined in this section will be duplicated and will have a comprehensive 
header indication which datebase were obtained, i.e, case report form (CRF) database and 
Independent review database. 
In this section additional tables and listings will be provided under request if clinical interest.” 
 
Section 13 Appendix IV. 
 
Original text: Figure 13.8 Boxplot of LVEF (baseline, nadir and last value available). 
Change to: Figure 13.8 Boxplot of LVEF (baseline, nadir and last value available). 


 
 
 
Section 14 Appendix V. 
  
Original text: Listing 14.1.17 Electrocardiogram*, 14.1.18 LVEF* and Listing 14.1.19 
Troponin I*  and (*) Also will be included information for the independent review mentioned 
in section 8.7 of this document. 
Change to: Listing 14.1.17 Electrocardiogram, 14.1.18 LVEF and Listing 14.1.19 Troponin I  
and (*) Also will be included information for the independent review mentioned in section 8.7 
of this document. 
 
Section 15 SAP Version History has been included in order to be consistent with the 
SAP amendment of this study and in accordance with PharmaMar’s SOP.