Conditions: Ewing Sarcoma Sarcoma
Keywords: Dose Limiting Toxicity Maximum Tolerated Dose Radiographic Response Time to Progression Bone Tumors
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 8
Healthy volunteers: no

Brief summary:
Background:

\- Mithramycin is a drug that was first tested as a cancer therapy in the 1960s. It acted against some forms of cancer, but was never accepted as a treatment. Research suggests that it may be useful against some solid tumors, particularly Ewing sarcoma. Researchers want to see if mithramycin can be used to treat solid tumors in children and adults. It will be tested in different groups of people, including those with a type of Ewing sarcoma that contains a chemical called Ewings sarcoma - friend leukemia integration 1 transcription factor (EWS-FLI1).

Objectives:

\- To see if mithramycin is safe and effective against solid tumors and Ewing sarcoma in children and adults.

Eligibility:

* Children and young adults between 1 and 17 years of age with solid tumors that have not responded to standard treatment.
* Adults at least 18 years of age with EWS-FLI1 Ewing sarcoma that has not responded to standard treatment.
* Children and young adults between 1 and 17 years of age with EWS-FLI1 Ewing sarcoma that has not responded to standard treatment.

Design:

* Participants will be screened with a physical exam and medical history. Blood and urine samples will be collected. Imaging studies and tumor tissue samples will be used to monitor the cancer before treatment. Individuals with solid brain tumors will not be eligible.
* Participants will receive mithramycin every day for 7 days, followed by 14 days without treatment. Each 28-day round of treatment is called a cycle.
* Treatment will be monitored with frequent blood tests and imaging studies.
* Participants will continue to take the drug for as long as the side effects are not severe and the tumor responds to treatment.

Detailed description:
BACKGROUND:

* Mithramycin, an anti-tumor antibiotic, underwent broad clinical evaluation in solid tumors and leukemias in the 1960s and demonstrated activity in some leukemias, lymphomas, and solid tumors. In particular, mithramycin was found to have activity against testicular cancers and was briefly used in the clinic for this tumor prior to the development of the currently used treatment regimen.
* The Ewing Sarcoma Family of Tumors (ESFT) is the second most common malignant bone tumor of childhood. There has been very little improvement in overall patient survival in past years, particularly for patients with high risk metastatic or relapsed disease. Therefore, there is a need for effective novel agents for the treatment of this disease.
* Multiple studies have shown that suppressing the expression of EWS-FLI1 effectively limits the tumorigenicity of ESFT cells. Laboratory studies have shown that mithramycin effectively suppresses the activity of EWS-FLI1 both in vitro and in vivo.

OBJECTIVES (PRIMARY):

* Phase I portion of this study is to: determine the tolerability, toxicity, and the recommended phase II dose of mithramycin in children and adolescents with refractory extracranial solid tumors.
* Phase II portion of this trial is to: determine the objective response rate (complete response (CR) and partial response (PR)) of Ewing sarcoma to mithramycin in children and adults using Response Evaluation Criteria in Solid Tumors (RECIST) criteria when administered at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated every 28 days until unacceptable toxicity or disease progression.
* Phase II portion of this trial to: evaluate if mithramycin inhibits NR0B1 in tumor tissue and determine changes in gene expression signature pre-treatment and at steady state on day +4 of treatment in patients greater than or equal to 18 years old with Ewing sarcoma and EWS/FLI1 fusion transcript with disease amenable to percutaneous biopsy.

ELIGIBILITY:

* Phase I Portion: children (greater than or equal to 12 months) and adolescents (less than or equal to 17 years) with recurrent or refractory extracranial solid tumors.
* Phase II Portion in adults: adults (greater than or equal to 18 years of age at enrollment) with recurrent or refractory measurable extracranial Ewing sarcoma and the EWS-FLI1 fusion transcript.
* Phase II Portion in children and adolescents: Once the adult dose is deemed safe, children

(greater than or equal to 12 months) and adolescents (less than or equal to 17 years) with recurrent or refractory measurable

extracranial Ewing sarcoma and the EWS-FLI1 fusion transcript will begin enrollment to the Phase II portion.

* Participants must meet safety laboratory criteria and prior therapy limitations.

DESIGN:

Phase I Portion: Mithramycin will be administered in escalating doses to children and adolescents intravenously over 6 hours once daily for 7 days to be repeated every 28 days until unacceptable toxicity or disease progression. The cohort at the recommended dose or maximum tolerated dose (MTD) will be expanded up to 12 patients, and attempts will be made to enroll 6 patients that are greater than or equal to 12 years of age and 6 patients that are \< 12 years of age to gain experience with a broad age range of patients. A maximum of 18 evaluable patients will be enrolled on the phase I portion.

* Phase II Portion: Using a Simon two stage design, mithramycin will be administered intravenously at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated every

  28 days until unacceptable toxicity or disease progression to children and adults with Ewing sarcoma with EWS-FLI1 fusion transcript. Up to 24 evaluable patients will be enrolled on the phase II portion.
* The Phase I and Phase II portions of the protocol will enroll patients simultaneously.

Arms:
arm 1: Dose Escalation Phase 9.0 mcg/kg.dose arm 2: Dose Escalation Phase 13.0 mcg/kg.dose arm 3: Dose Escalation Phase 17.5 mcg/kg.dose arm 4: Expansion phase 17.5 mcg/kg.dose

Interventions: intervention 1: Mithramycin
Intervention descriptions: intervention 1: Phase I Portion: Mithramycin will be administered in escalating doses to children and adolescents intravenously over 6 hours once daily for 7 days to be repeated every 28 days until unacceptable toxicity or disease progression. If maximum tolerated dose (MTD) in this Phase differs from the recommended adult dose for Phase II, the protocol will be amended. Using a Simon two stage design, mithramycin will be administered intravenously at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated every 28 days until unacceptable toxicity or disease progression to children and adults with Ewing sarcoma with Ewings sarcoma - friend leukemia integration 1 transcription factor (EWS-FLI1) fusion transcript Both Phases will enroll patients simultaneously. Phase II Portion: mithramycin will be administered intravenously at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated every 28 days until unacceptable toxicity or disease progression to children and adults.

Protocol document:
ICF_000.pdf: 
MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
•
Adult Patient or
•
Parent, for Minor Patient
INSTITUTE: 
National Cancer Institute 
STUDY NUMBER: 
12-C-0135
PRINCIPAL INVESTIGATOR: 
Brigitte Widemann, M.D. 
STUDY TITLE: 
Phase I/II Trial of Mithramycin in Children and Adults with Refractory Extracranial 
Solid Tumors (Phase I) or Ewing Sarcoma and EWS-FLI1 Fusion Transcript (Phase 
II) 
Continuing Review Approved by the IRB on 01/12/15 
Amendment Approved by the IRB on 12/24/14 (E) 
Date posted to web:  01/22/15 
Phase I Portion 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
•
Adult Patient or
•
Parent, for Minor Patient
NIH-2514-1 (07-09)
P.A.:  09-25-0099
File in Section 4:  Protocol Consent (1)
INTRODUCTION 
We invite you to take part in a research study at the National Institutes of Health (NIH). 
First, we want you to know that: 
Taking part in NIH research is entirely voluntary. 
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  However, 
to receive care at the NIH, you must be taking part in a study or be under evaluation for 
study participation. 
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If you 
have such beliefs, please discuss them with your NIH doctors or research team before you agree 
to the study. 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with anyone at NIH, or with family, 
friends or your personal physician or other health professional.   
If you are signing for a minor child, “you” refers to “your child” throughout the consent 
document. 
Why is this study being done? 
The purpose of this study is to test the safety and tolerability of a drug called mithramycin in 
children and adolescents with solid tumors.  Once a safe dose of mithramycin is determined, we 
will test whether mithramycin can shrink tumors in children and young adults with Ewing 
sarcoma who have a specific molecular change in their tumor.  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 2 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Mithramycin is an experimental drug that was tested as a cancer therapy in the 1960’s and was 
found to have activity against some forms of cancer, but was never broadly accepted as a 
treatment.  Mithramycin is considered experimental because it has not been approved by the US 
Food and Drug Administration (FDA) for treatment of your type of cancer.  Because of its prior 
activity, researchers have continued laboratory research on the ability of mithramycin to fight 
specific cancers.  In the laboratory mithramycin is active against a variety of childhood solid 
tumors, and in particular Ewing sarcoma.  Prior research studies did not provide doctors with the 
highest safe dose of mithramycin in children, so in the first phase (Phase I) of this study, we will 
determine the highest safe dose.   
Once the highest safe dose is established, this dose will be used to test if mithramycin is effective 
in shrinking tumors in individuals with Ewing sarcoma in the second phase of the study.  This 
informed consent is for patients participating in the first phase of the study.  
Why are you being asked to take part in this study? 
You are being invited to participate in this study because you have advanced cancer that is not 
responding to known effective therapies.  
How many people will take part in this Study?  
Up to 18 children and adolescents will take part in phase I of the study. 
Description of Research Study 
What will happen if you take part in this research study?   
Before you begin the study 
You will need to have the following exams, tests or procedures to find out if you can be in the 
study.  These exams, tests or procedures are part of regular cancer care and may be done even if 
you do not join the study.  If you have had some of them recently, they may not need to be 
repeated.  This will be up to your study doctor.   
• A medical history 
• Physical exam 
• Vital signs (blood pressure, pulse, temperature) 
• Blood tests 
• Urine tests 
• Pregnancy test (if you are a woman who could have children) 
• Echocardiogram or MUGA and an EKG,  tests that checks the function of 
your heart 
• We will also do whatever X-rays, CT scans, or other tests are needed to check 
your tumor 
• Review of your tumor tissue to confirm your diagnosis 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 3 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
During the study 
If the exams, tests and procedures show that you can be in the study, and you choose to take part, 
you will be admitted to the hospital at NIH and given mithramycin through an IV (intravenous – 
small plastic tube put in a vein in your arm or neck) over about 6 hours.  You will receive 
mithramycin every day for 7 days.  If you do not have unacceptable side effects or worsening of 
your cancer, this treatment can be repeated every 28 days.  This is called a cycle.  You will 
receive the first cycle of mithramycin as an inpatient in the hospital so that we can monitor you 
carefully for side effects and treat them.  You will also be given dexamethasone every day in 
order to prevent mithramycin liver toxicity. You may receive subsequent cycles as an outpatient 
if your doctor believes this is safe for you.  
The dose for the first children enrolled on the study will be based on the side effects seen in 
adults.  Between 3 and 6 children will receive mithramycin at each dose.  There will be two 
doses tested in this study.  If the side effects are not too severe, the next group of children will 
receive a higher dose.  This is called ‘dose escalation’.  Whatever dose you start at, your dose 
will not be increased.  If you have bad side effects, the drug will be stopped or delayed.  You will 
be given medicines to help treat any side effects that you might have. 
While you are receiving cycles of mithramycin, you will be watched closely for any untoward 
effects.  You will need to have the following procedures and tests: 
• Physical exam every week 
• Blood tests daily during mithramycin treatment and then twice a week for 
the rest of the cycle 
Between doses we will ask you to fill out forms to help keep track of any symptoms you may 
experience and any medications you may take. 
Scans and x-rays to evaluate your tumor will be done after every 2 cycles (about 2 months, 4 
months, etc).  
RESEARCH STUDY TESTS AND PROCEDURES 
We would also like to do some extra research tests.  These tests will help us learn more about 
mithramycin and may help children who receive this drug in the future.  The information learned 
from these tests will not change the way you are treated, and the results of these tests will not be 
returned to you.  You can still be a part of the main study even if you say ‘no’ to taking part in 
any of the optional biology studies.  The maximum amount of blood taken from you is based on 
your age and will not be more than the blood volume limit set for research by the NIH.  We will 
not draw more than 1 teaspoon of blood for every kilogram (2.2 pounds) of your body weight in 
a single day and not more than 2 teaspoons of blood for every kilogram (2.2 pounds) of your 
body weight in an 8 week period.  For example, if you weigh 85 pounds (weight of some 12 year 
olds), your weight in kilograms is about 38 kg; therefore we would not draw more 38 teaspoons 
(12.5 tablespoons) in one day and not more than 76 teaspoons (or 25 tablespoons) of blood in an 
8 week period. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 4 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
A check box for your decision about taking part in each optional test is provided at the end of 
this consent form.  The following is a description of each of the optional tests. 
Pharmacokinetic Studies (PKs)  
We want to find out how mithramycin is processed and then cleared from the body.  These tests 
are called pharmacokinetic (PK) tests.  They will measure the amount of mithramycin in the 
blood at different time points during treatment.  
If you agree, we would like to obtain blood samples from you.  These samples are about 3 mL 
(about ½  teaspoon) of blood each and will be taken before the drug begins, and at the middle 
and end of the first mithramycin infusion (on day 1), and then just before and at the completion 
of the mithramycin infusion on days 2, 4 and day 7 of your first cycle.  A sample will also be 
obtained 24 and 48 hours after the day 7 dose.  A total of 24 mL of blood will be drawn 
(approximately 4-5 teaspoons) will be drawn for PK studies.  
Please read the sentence below and think about your choice.  After reading the sentence, circle 
and initial the answer that is right for you. 
I agree to have the pharmacokinetic studies performed in this study. 
 
Yes              No               Initials_________ 
 
Pharmacodynamic studies 
We would also like to take a small amount of blood for research tests called pharmacodynamic 
studies (PD).  These tests will help us learn more about mithramycin and may help children who 
receive this drug in the future.  The information learned would not change the way you are 
treated, and the results of these tests will not be given to you.  A total of 1 mL of blood will be 
drawn (approximately 1/5 teaspoon) at the following time points: before the first dose of 
mithramycin, before the dose at the start of every other cycle.  Prior to this blood sample you will 
need to not eat or drink anything for 10-12 hours.  You should also avoid antacids or any 
medication that helps you have a bowel movement like Reglan, Metamucil, Miralax, for 48 
hours prior to the blood test.  
The amount of blood we are drawing for research studies is safe to draw even from small 
children. 
Please read the sentence below and think about your choice.  After reading the sentence, circle 
and initial the answer that is right for you. 
I agree to have research blood drawn for pharmacodynamic studies performed in this study. 
 
Yes              No               Initials_________ 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 5 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Archival Tumor Tissue 
One of the tests we would like to perform is done on tissue previously obtained from your tumor.  
It is called immunohistochemistry or IHC and is used to evaluate for NR0B1 expression.  
NR0B1 is a gene which gives directions (or encodes) for a protein normally found in the body.  
Mutations or changes in the NR0B1 gene may result in mishappen or nonfunctional proteins, 
which may lead to certain disorders.  We plan to test tumor tissue that was previously obtained 
during surgery or a biopsy.  We will not ask you to have a biopsy to do this test.  This is a 
research test and the information learned will not change the way you are treated, and the results 
of these tests will not be given to you. 
I agree to allow previously obtained tumor tissue to be used for research tests in this study. 
 
Yes              No               Initials_________ 
Review of Scans 
Copies of the scans used to diagnose your tumors and scans taken to see if your tumor is 
responding to treatment will be sent to a central review center as part of our quality control 
program.   
We will also have a radiologist review the scans of your tumor to study a new method of 
measuring the volume of tumors in patients with Ewing Sarcoma.  Currently we measure tumor 
size by two common methods: one method measures the longest length of the tumor, and the 
second method measures the length and the width, or two dimensions of the tumor.  In the 
proposed scan review, we will compare these two methods with a new method that measures the 
volume, or 3 dimensions, of the tumor to see which method gives us the best information for 
determining tumor size.  
How long will I be in the study? 
You may be in the study until one of the following occur:   
• Your tumor gets worse 
• The side effects of mithramycin are too harmful for you 
• You need a treatment that is not allowed on this study 
• You are not able to follow study-related treatment instructions 
• New information becomes available that suggests this is not the best treatment for you 
• The study is not in your best interest 
• The study is stopped 
 
Your doctor may decide to take you off the study drug if any of these occur; you will be told the 
reason you are being taken off the mithramycin. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 6 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
When you are finished taking the drugs (treatment) 
Once the mithramycin is stopped, you will need to come back to NIH for evaluation (physical 
exam and blood work) until you recover from any side effects, and at least 30 days after the last 
dose of mithramycin.  Afterwards, the study doctor will refer you back to your local health care 
provider. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 7 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Study Chart 
Cycle 1 
Subsequent Cycles 
DAY 
WHAT YOU DO 
DAY 
WHAT YOU DO 
Before 
starting 
study 
Come into the clinic and do the 
following: 
• Routine blood tests 
• Urine tests 
• Pregnancy test if you are able to 
have children 
• Physical exam by your doctor 
• MRI, PET or CT scan 
• EKG 
• Get a disease evaluation that will be 
done by your doctor. Depending on 
the results of this evaluation, your 
doctor will tell you whether or not 
you may begin this study. 
 
Day 1 
Receive oral/IV dexamethasone (12 
hours before study drug) 
Come to NIH hospital and do the 
following: 
• Routine blood tests 
• Physical exam  
• EKG 
• Prior to first dose of study drug 
receive oral/IV dexamethasone 
• Get the study drug mithramycin  
• Twice daily for 24 hours after the 
last dose of study drug receive 
oral/IV dexamethasone 
• Blood drawn before and after 
mithramycin infusion for PKs  
• Blood drawn for PD studies if you 
have Ewing sarcoma (Phase I and II) 
Day 1 
Come to NIH hospital and do 
the following: 
• Routine blood tests 
• Physical exam  
• EKG 
• Get the study drug 
mithramycin 
• Twice daily for 24 hours 
after the last dose of 
study drug receive 
oral/IV dexamethasone 
• Blood drawn for PD 
studies (Phase I and II) 
on Cycles 3, 5, 7, 9, 11, 
and 13. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 8 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Days 2-
7   
• Routine blood tests 
• Get the study drug mithramycin 
• Twice daily for 24 hours after the 
last dose of study drug receive 
oral/IV dexamethasone 
• EKG on days 4 & 7  
 
Days 2-
7  
• Routine blood tests 
(cycles 2, 3) 
• Routine blood tests twice 
weekly (cycles 4 to the 
end) 
• Get the study drug 
mithramycin 
• Twice daily for 24 hours 
after the last dose of study 
drug receive oral/IV 
dexamethasone 
• EKG on day 7 
Days 1, 
2, 4, 7, 
8, 9 
Blood drawn for mithramycin PKs  
 
Day 
9 
to 28 
Come into the clinic and do the 
following: 
• Routine blood tests twice per week 
• Physical exam by your doctor every 
week 
Day 9 to 
28 
Come into the clinic and do 
the following: 
• Routine blood tests twice 
per week (cycles 2 and 3) 
• Routine blood tests 
weekly for cycles 4 to the 
end 
• Physical exam by your 
doctor every week 
 
Every 2 
cycles 
Come into the clinic and do 
the following: 
• Routine blood tests 
• Physical exam by your 
doctor 
• MRI or CT scan 
• Disease evaluation that 
will be done by your 
doctor. Depending on the 
results of this evaluation, 
your doctor will tell you 
whether or not you may 
continue the next cycle 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 9 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Birth Control 
Reproductive risks:  You should not become pregnant or father a baby while on this study 
because the drugs in this study can affect an unborn baby.  If you become pregnant on the study, 
you will be taken off of mithramycin immediately.  Further, if the pregnancy is taken to term, the 
outcome will also be recorded in study records.  Women should not breastfeed a baby while on 
this study.  It is important you understand that you need to use birth control while on this study 
and for at least two months after finishing treatment with mithramycin.  Check with your study 
doctor about what kind of birth control methods to use and how long to use them.  Some 
methods might not be approved for use in this study. 
Other medications 
There are several medications, such as aspirin or ibuprofen, which are not allowed during 
participation in this study, as these medications may increase the risk for mithramycin side 
effects, such as bleeding.  It is very important that you contact your doctor prior to starting a new 
medicine (including herbal or over the counter medications) to check if it is OK to take it.  The 
study team will provide you and your home doctor with a list of medicines, which you should not 
take.  
Alternative Approaches or Treatments 
What other choices do I have if I do not take part in this study?  
 
Instead of being in this study, you have these options: 
• Getting treatment or care for your cancer without being in a study 
• Taking part in another study 
• Getting comfort care, also called palliative care.  This type of care helps reduce pain, 
tiredness, appetite problems and other problems caused by the cancer.  It does not treat 
the cancer directly.  Instead, it tries to improve how you feel.  Comfort care tries to keep 
you as active and comfortable as possible. 
Please talk to your doctor about these and other options. 
Risks or Discomforts of Participation 
What side effects or risks can I expect from being in this study?  
You may have side effects while on the study.  Everyone taking part in the study will be watched 
carefully for any side effects.  However, doctors don’t know all the side effects that may happen.  
Side effects may be mild or very serious. Your health care team may give you medicines to help 
lessen side effects.  Many side effects go away soon after you stop taking the mithramycin.  In 
some cases, side effects can be serious, long lasting, or may never go away.  Rarely there is a 
risk of death from a serious side effect.  We will tell you if we learn any new information that 
may affect your health, welfare, or decision to stay in this study.  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 10 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
You should talk to your study doctor about any side effects that you have while taking part in the 
study.    
Side Effects from Mithramycin 
Likely 
• Decreased blood level of calcium 
• Nausea or vomiting 
• Diarrhea 
• Loss of appetite 
• Mouth sores  
• Increase blood levels of liver enzymes, which may indicate damage to the liver (which 
may be severe) 
• Increase in LDH, which is an indicator of damage to your cells. 
Less Likely 
• Bleeding including nose bleed 
• Fever 
• Drowsiness 
• Weakness 
• Tiredness 
• Pain, redness, soreness or swelling of the area where the IV goes in the skin 
• Skin changes, blushing of the face   
• Headache 
• Mental depression, restlessness, irritability 
• Decreased platelets (the blood cell that helps blood clot) 
• Decreased red blood cells (the blood cells that carry oxygen) 
• Abnormal levels of white blood cells (the cells that prevent infection) 
• Muscle or stomach cramps, possibly due to a low blood level of calcium  
• Abnormal levels of blood elements including phosphorus or potassium. 
• Increased blood levels of kidney function tests, such as creatinine, urea or protein 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 11 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Rare but Serious 
• Bleeding that may require a transfusion of blood or blood products.  Bleeding may result 
in bloody or black stools or vomiting blood, or blood nose, or small broken blood vessels 
under the skin. 
• A significant rash called toxic epidermal necrolysis characterized by widespread and 
severe skin irritation has been reported with mithramycin. 
While receiving mithramycin, you should avoid any drugs that contain salicylates, a component 
of aspirin, such as Aspirin, Bufferin, Ascriptin, Aspergum, Anacin, and some Alka-Seltzer 
products and some cold preparations, as they may increase the risk of bleeding. 
When the WBC is low (neutropenia), fever may be a sign of significant infection.  When fever 
and neutropenia is present, hospitalization for administration of intravenous antibiotics may be 
necessary. 
Because mithramycin may decrease the production of red cells (the part of the blood that carries 
oxygen and gives energy) and platelets (the part of the blood that helps prevent bleeding), it is 
possible that you will require transfusions with red cells or platelets.  When these transfusions 
are required will vary from participant to participant and can be discussed with your doctor.  
Most transfusions have no side effects.  However, you may have an allergic reaction (a rash, 
hives or very rarely, difficulty breathing that can be so severe as to result in death).  In addition, 
although the blood bank that services our patients is excellent, there is at least a theoretic 
possibility of infections such as AIDS or hepatitis being transmitted by blood.  The blood bank is 
very good at screening for these diseases and the chance of these problems is very small.  A 
separate informed consent would be obtained should you need a blood transfusion. 
Side Effects from Dexamethasone 
• High blood pressure 
• High Blood glucose 
• Stomach upset, 
• Burning in stomach from excess stomach acid 
• Increased risk of infection 
• Increased appetite  
• Weight gain 
• Thinning of bones  
• Increase pressure in eyes 
• Personality changes, e.g., irritability, euphoria, mania 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 12 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• Feelings of depression 
• Pulmonary tuberculosis 
• Vision problems 
• Acne, allergic dermatitis, dry scaly skin 
• Destruction of bone tissue 
Potential Benefits of Participation 
Are there benefits to taking part in this study?  
The potential benefit of the treatment with mithramycin is that it may cause your cancer to stop 
growing or to shrink for a period of time.  It may lessen the symptoms, such as pain, that are 
caused by the cancer.  It is extremely unlikely that this treatment will cure your cancer.  Because 
there is not much information about the mithramycin effect on cancers in humans, we do not 
know if you will benefit from taking part in this study.  Information learned from this study may 
help future patients with cancer. 
Research Subject’s Rights 
What are the costs of taking part in this study?  
If you choose to take part in the study, the following will apply, in keeping with the NIH policy: 
• 
You will receive study treatment at no charge to you. This may include 
surgery, medicines, laboratory testing, x-rays or scans done at the 
Clinical Center, National Institutes of Health (NIH), or arranged for you 
by the research team to be done outside the Clinical Center, NIH if the 
study related treatment is not available at the NIH.  
• 
There are limited funds available to cover the cost of some tests and 
procedures performed outside the Clinical Center, NIH.  You may have 
to pay for these costs even if they are not covered by your insurance 
company. 
• 
Medicines that are not part of the study treatment will not be provided or 
paid for by the Clinical Center, NIH. 
• 
Once you have completed taking part in the study, medical care will no 
longer be provided by the Clinical Center, NIH. 
Will my medical information be kept private?  
We will do our best to make sure that the personal information in your medical record will be 
kept private.  However, we cannot guarantee total privacy.  Your personal information may be 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 13 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
given out if required by law.  If information from this study is published or presented at scientific 
meetings, your name and other personal information will not be used.  
Organizations that may look at and/or copy your medical records for research, quality assurance, 
and data analysis include: 
• Representatives of the  National Cancer Institute (NCI) and other government agencies, 
like the Food and Drug Administration (FDA), involved in overseeing research 
• The Institutional Review Board (IRB) of this hospital 
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law.  This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results.  You can search this Web site at any time.” 
 
Stopping Therapy  
You can stop taking part in the study at any time.  However, if you decide to stop taking part in 
the study, we would like you to talk to the study doctor and your regular doctor first. 
Conflict of Interest 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of 
interest. 
 
The 
following 
link 
contains 
details 
on 
this 
process 
http://ethics.od.nih.gov/procedures/COI-Protocol-Review-Guide.pdf.  You may ask your 
research team for a copy of the Protocol Review Guide or for more information.  Members of the 
research team who do not work for NIH are expected to follow these guidelines but they do not 
need to report their personal finances to the NIH. 
 
Members of the research team working on this study may have up to $15,000 of stock in the 
companies that make products used in this study.  This is allowed under federal rules and is not a 
conflict of interest.  
 
Optional Studies  
We would like to keep some of the specimens and data that are collected for future research.  
These specimens and data will be identified by a number and not your name.  The use of your 
specimens and data will be for research purposes only and will not benefit you.  It is also 
possible that the stored specimens and/or data may never be used.  Results of research done on 
your specimens and data will not be available to you or your doctor.  It might help people who 
have cancer and other diseases in the future.   
 
If you decide now that your specimens and data can be kept for research, you can change your 
mind at any time. Just contact us and let us know that you do not want us to use your specimens 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0135 
CONTINUATION: page 14 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
and/or data.  Then any specimens that remain will be destroyed, and your data will not be used 
for future research.  
Please read each sentence below and think about your choice. After reading each sentence, circle 
and initial the answer that is right for you.  No matter what you decide to do, it will not affect 
your care. 
 
1. My specimens and data may be kept for use in research to learn about, prevent, or treat cancer.  
 
Yes              No               Initials_________ 
 
2. My specimens and data may be kept for use in research to learn about, prevent or treat other 
health problems (for example: diabetes, Alzheimer's disease, or heart disease).  
 
Yes              No               Initials_________ 
 
3. Someone may contact me in the future to ask permission to use my specimens and/or data in 
new research not included in this consent.  
 
Yes              No               Initials_________ 
 
 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 15 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
(Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
OTHER PERTINENT INFORMATION 
1. 
Confidentiality.  When results of an NIH research study are reported in medical journals 
or at scientific meetings, the people who take part are not named and identified.  In most cases, 
the NIH will not release any information about your research involvement without your written 
permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This 
information might affect (either favorably or unfavorably) the willingness of the insurance 
company to sell you insurance. 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you 
should know that the Act allows release of some information from your medical record without 
your permission, for example, if it is required by the Food and Drug Administration (FDA), 
members of Congress, law enforcement officials, or authorized hospital accreditation 
organizations. 
2. 
Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-
term medical care for any injury resulting from your participation in research here.  In general, 
no long-term medical care or financial compensation for research-related injuries will be 
provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  
However, you have the right to pursue legal remedy if you believe that your injury justifies such 
action. 
3. 
Payments.  The amount of payment to research volunteers is guided by the National 
Institutes of Health policies.  In general, patients are not paid for taking part in research studies at 
the National Institutes of Health.  Reimbursement of travel and subsistence will be offered 
consistent with NIH guidelines. 
4. 
Problems or Questions.  If you have any problems or questions about this study, or 
about your rights as a research participant, or about any research-related injury, contact the 
Principal Investigator, Dr. Brigitte Widemann at Telephone: 301-496-7387, or Ms. Donna 
Bernstein, Building 10, Room13C101, research nurse for the study, Telephone: 301-435-7804 . 
You may also call the Clinical Center Patient Representative at (301) 496-2626. If you have any 
questions about the use of your specimens and data for future research studies, you may also 
contact the Office of the Clinical Director, Telephone:  301-496-4251. 
5. Consent Document.  Please keep a copy of this document in case you want to read it again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 16 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
(Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
COMPLETE APPROPRIATE ITEM(S) BELOW: 
A. 
Adult Patient’s Consent B. 
Parent’s Permission for Minor Patient. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby consent to 
take part in this study. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby give 
permission for my child to take part in this 
study.   
(Attach NIH 2514-2, Minor’s Assent, if 
applicable.) 
 
 
 
 
Signature of Adult Patient/ 
Legal Representative 
Date 
Signature of Parent(s)/ Guardian Date 
 
Print Name 
 
Print Name 
C.  Child’s Verbal Assent (If Applicable) 
The information in the above consent was described to my child and my child agrees to 
participate in the study. 
 
 
 
Signature of Parent(s)/Guardian 
Date 
Print Name 
THIS CONSENT DOCUMENT HAS BEEN APPROVED FOR USE  
FROM JANUARY 12, 2015 THROUGH JANUARY 11, 2016. 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
Print Name 
 
Print Name 
 

ICF_001.pdf: 
MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
INSTITUTE: 
National Cancer Institute 
STUDY NUMBER: 
12-C-0135 
PRINCIPAL INVESTIGATOR: 
Brigitte Widemann, M.D. 
STUDY TITLE:  
Phase I/II Trial of Mithramycin in Children and Adults with Refractory Extracranial 
Solid Tumors (Phase I) or Ewing Sarcoma and EWS-FLI1 Fusion Transcript (Phase 
II) 
Continuing Review Approved by the IRB on 01/12/15 
Amendment Approved by the IRB on 12/24/14 (E) 
Date posted to web:  01/22/15 
Phase II portion 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent (2) 
 
 
INTRODUCTION 
We invite you to take part in a research study at the National Institutes of Health (NIH). 
First, we want you to know that: 
Taking part in NIH research is entirely voluntary.  
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  However, 
to receive care at the NIH, you must be taking part in a study or be under evaluation for 
study participation. 
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If you 
have such beliefs, please discuss them with your NIH doctors or research team before you agree 
to the study. 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with anyone at NIH, or with family, 
friends or your personal physician or other health professional.   
If you are signing for a minor child, “you” refers to “your child” throughout the consent 
document. 
Why is this study being done?  
The purpose of this study is to test whether mithramycin can shrink tumors in children and young 
adults with Ewing sarcoma who have a specific molecular change in their tumor.  
Mithramycin is an experimental drug that was tested as a cancer therapy in the 1960’s and was 
found to have activity against some forms of cancer, but was never broadly accepted as a 
treatment.  Mithramycin is considered experimental because it has not been approved by the US 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 2 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Food and Drug Administration (FDA) for treatment of your type of cancer.  Because of its prior 
activity, researchers have continued laboratory research on the ability of mithramycin to fight 
specific cancers.  In the laboratory mithramycin is active against a variety of childhood solid 
tumors, and in particular Ewing sarcoma.   
The dose of mithramycin you will be given is the same dose that was shown to be relatively safe 
in previous studies in adults.  We will test whether mithramycin is effective in shrinking tumors 
in individuals with Ewing sarcoma.  
 
Why are you being asked to take part in this study? 
You are being invited to participate in this study because you have advanced cancer that is not 
responding to known effective therapies.  
How many people will take part in this study?  
A maximum of 26 people with Ewing Sarcoma will take part in this study. 
Description of Research Study 
What will happen if you take part in this research study?   
Before you begin the study 
You will need to have the following exams, tests or procedures to find out if you can be in the 
study.  These exams, tests or procedures are part of regular cancer care and may be done even if 
you do not join the study.  If you have had some of them recently, they may not need to be 
repeated.  This will be up to your study doctor.   
• A medical history 
• Physical exam 
• Vital signs (blood pressure, pulse, temperature) 
• Blood tests 
• Urine tests 
• Pregnancy test (if you are a woman who could have children) 
• Echocardiogram or MUGA and an EKG, tests that checks the function of your 
heart 
• We will also do whatever X-rays, CT scans, or other tests are needed to check 
your tumor 
• Review of your tumor tissue to confirm your diagnosis 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 3 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
During the study 
If the exams, tests and procedures show that you can be in the study, and you choose to take part, 
you will be admitted to the hospital at NIH and given mithramycin through an IV (intravenous – 
small plastic tube put in a vein in your arm or neck) over about 6 hours.  You will receive 
mithramycin every day for 7 days.  If you do not have unacceptable side effects or worsening of 
your cancer, this treatment can be repeated every 28 days.  This is called a cycle.  You will 
receive the first cycle of mithramycin as an inpatient in the hospital so that we can monitor you 
carefully for side effects and treat them.  You may receive subsequent cycles as an outpatient if 
your doctor believes this is safe for you.  
If you have bad side effects, the drug will be stopped, delayed, or given at a reduced dose.  You 
will be given medicines to help treat any side effects that you might have. 
While you are receiving cycles of mithramycin, you will be watched closely for any untoward 
effects.  You will need to have the following procedures and tests: 
• Physical exam every week 
• Blood tests daily during mithramycin treatment and then twice a week for 
the rest of the cycle. 
Between doses we will ask you to fill out forms to help keep track of any symptoms you may 
experience and any medications you may take. 
Scans and x-rays to evaluate your tumor will be done after every 2 cycles (about 2 months, 4 
months, etc).  
 
RESEARCH STUDY TESTS AND PROCEDURES 
We would also like to do some extra research tests.  These tests will help us learn more about 
mithramycin and may help children who receive this drug in the future.  The information learned 
from these tests will not change the way you are treated, and the results of these tests will not be 
returned to you.  You can still be a part of the main study even if you say ‘no’ to taking part in 
any of the optional biology studies.  The maximum amount of blood taken from you is based on 
your age and will not be more than the blood volume limit set for research by the NIH.  In adults 
that limit is 37 tablespoons in an 8 week period.  If you are under age 18, we will not draw more 
than 1 teaspoon of blood for every kilogram (2.2 pounds) of your body weight in a single day 
and not more than 2 teaspoons of blood for every kilogram (2.2 pounds) of your body weight in 
an 8 week period.  For example, if you weigh 85 pounds (weight of some 12 year olds), your 
weight in kilograms is about 38 kg; therefore we would not draw more 38 teaspoons (12.5 
tablespoons) in one day and not more than 76 teaspoons (or 25 tablespoons) of blood in an 8 
week period. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 4 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
A check box for your decision about taking part in each optional test is provided at the end of 
this consent form.  The following is a description of each of the optional tests. 
Pharmacokinetic Studies (PKs)  
We want to find out how mithramycin is processed and then cleared from the body.  These tests 
are called pharmacokinetic (PK) tests.  They will measure the amount of mithramycin in the 
blood at different time points during treatment.  
If you agree, we would like to obtain blood samples from you.  These samples are about 3 mL 
(about ½  teaspoon) of blood each and will be taken before the drug begins, and at the middle 
and end of the first mithramycin infusion (on day 1), and then just before and at the completion 
of the mithramycin infusion on days 2, 4 and day 7 of your first cycle.  A sample will also be 
obtained 24 and 48 hours after the day 7 dose.  A total of 24 mL of blood will be drawn 
(approximately 4-5 teaspoons) will be drawn for PK studies.  
Please read the sentence below and think about your choice.  After reading the sentence, circle 
and initial the answer that is right for you. 
I agree to have the pharmacokinetic studies performed in this study. 
 
Yes              No               Initials_________ 
Pharmacodynamic studies 
We would also like to take a small amount of blood for research tests called pharmacodynamic 
studies (PD).  These tests will help us learn more about mithramycin and may help children who 
receive this drug in the future.  The information learned would not change the way you are 
treated, and the results of these tests will not be given to you.  A total of 1 mL of blood will be 
drawn (approximately 1/5 teaspoon) at the following time points: before the first dose of 
mithramycin, before the dose at the start of every other cycle.  Prior to this blood sample you will 
need to not eat or drink anything for 10-12 hours.  You should also avoid antacids or any 
medication that helps you have a bowel movement like Reglan, Metamucil, Miralax, for 48 
hours prior to the blood test.  
The amount of blood we are drawing for research studies is safe to draw even from small 
children. 
Please read the sentence below and think about your choice.  After reading the sentence, circle 
and initial the answer that is right for you. 
I agree to have the pharmacodynamic studies performed in this study. 
 
Yes              No               Initials_________ 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 5 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Biopsy of Tumor Tissue 
If you are 18 years of age and older and have Ewing sarcoma and a small sample of tumor tissue 
can be easily obtained, you will be required to have a biopsy of tumor tissue in order to 
participate in the study.  A biopsy will be done prior to starting treatment and then after you have 
been taking the mithramycin for 4 days (+/- 1) on cycle 1 only, to evaluate the effects of this 
drug on your tumor tissue.  Biopsies are a very important part of this trial and are done for 
research purposes.  Willingness to undergo tumor biopsies is required for taking part in this 
study.  No more than two biopsy procedures will be performed during the study.  After the first 
biopsy, if you decide not to have further biopsies, you will still receive study drugs and have 
other tests that are part of the study.  You will be asked to sign a separate consent form for each 
biopsy procedure. 
Trained personnel will perform these biopsies.  If any complication occurs, we will offer medical 
care.  If upon attempting the first biopsy procedure, no tissue can be obtained or it has caused 
you harm, the second biopsy procedure will not be done.  After you are enrolled in this study, if 
for any reason the biopsies cannot be done safely, you can still receive the study drugs but the 
biopsies will not be performed. 
Biopsies will be done using a small bore (thin) needle under imaging guidance (CT, MRI, or 
ultrasound depending upon which provides the best picture for the interventional radiologist 
performing the biopsy).  Imaging helps the specialized radiologist know that the needle has been 
placed into the tumor mass.  Typical risks of such procedures include, but are not limited to, 
bleeding, infection, pain, and scarring.  You will be counseled in more detail about the 
procedure, and you will be asked to sign a separate consent form that will describe the procedure 
and its risks at that time.  Your safety is the most important thing at all times. 
The biopsy to be performed is only for research purposes and will not benefit you.  It might help 
other people in the future.  Even if you sign "yes" to have the biopsy, you can change your mind 
at any time.  Please read the sentence below and think about your choice.  After reading the 
sentence, circle and initial the answer that is right for you. 
I agree to have the tumor biopsy for the research tests in this study. 
 
Yes              No               Initials_________ 
Archival Tumor Tissue 
One of the tests we would like to perform is done on tissue previously obtained from your tumor.  
It is called immunohistochemistry or IHC and is used to evaluate for NR0B1 expression.  
NR0B1 is a gene which gives directions (or encodes) for a protein normally found in the body.  
Mutations or changes in the NR0B1 gene may result in mishappen or nonfunctional proteins, 
which may lead to certain disorders.  We plan to test tumor tissue that was previously obtained 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 6 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
during surgery or a biopsy.  We will not ask you to have a biopsy to do this test.  This is a 
research test and the information learned will not change the way you are treated, and the results 
of these tests will not be given to you. 
I agree to allow previously obtained tumor tissue to be used for research tests in this study. 
 
Yes              No               Initials_________ 
How long will I be in the study? 
You may be in the study until one of the following occurs.   
• Your tumor gets worse 
• The side effects of mithramycin are too harmful for you 
• You need a treatment that is not allowed on this study 
• You are not able to follow study-related treatment instructions 
• New information becomes available that suggests this is not the best treatment for you 
• The study is not in your best interest 
• The study is stopped 
Your doctor may decide to take you off study if any of these occur; you will be told the reason 
you are being taken off the mithramycin. 
When you are finished taking the drugs (treatment) 
Once the mithramycin is stopped, you will need to come back to NIH for evaluation (physical 
exam and blood work) until you recover from any side effects and at least 30 days after the last 
dose of mithramycin.  Afterwards, the study doctor will refer you back to your local health care 
provider. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 7 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Study Chart 
Cycle 1 
Subsequent Cycles 
DAY 
WHAT YOU DO 
DAY 
WHAT YOU DO 
Before 
starting 
study 
Come into the clinic and do the 
following: 
• Routine blood tests 
• Urine tests 
• Pregnancy test if you are female 
and able to have children 
• Physical exam by your doctor 
• MRI, PET or CT scan 
• EKG 
• Get a disease evaluation that will 
be done by your doctor. Depending 
on the results of this evaluation, 
your doctor will tell you whether 
or not you may begin this study. 
 
Day 1 
Receive oral/IV dexamethasone (12 
hours before study drug) 
Come to NIH hospital and do the 
following: 
• Routine blood tests 
• Physical exam  
• EKG 
• Prior to first dose of study drug 
receive oral/IV dexamethasone 
• Get the study drug mithramycin 
• Twice daily for 24 hours after the 
last dose of study drug receive 
oral/IV dexamethasone 
• Blood drawn before and after 
mithramycin infusion for PKs 
• Blood drawn for PD studies if you 
have Ewing sarcoma  
• Mandatory biopsy for patients with 
Ewing Sarcoma who are 18 years 
old or older before starting drug 
Day 1 
Come to NIH hospital and do the 
following: 
• Routine blood tests 
• Physical exam 
• EKG  
• Get the study drug 
mithramycin 
• Twice daily for 24 hours 
after the last dose of study 
drug receive oral/IV 
dexamethasone 
• Blood drawn for PD 
studies on Cycles 3, 5, 7, 9, 
11, and 13. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 8 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Days 1, 
2, 4, 7, 
8, 9 
Blood drawn for mithramycin PKs  
 
 
About 
Day 4 
• Mandatory second biopsy  
• EKG 
 
 
 
Days 2-
7  
• Routine blood tests 
• Get the study drug mithramycin 
• Twice daily for 24 hours after the 
last dose of study drug receive 
oral/IV dexamethasone 
• EKG on day 7  
 
Days 
2-7  
• Routine blood tests (cycles 2, 
3) 
• Routine blood tests twice 
weekly only (cycles 4 to the 
end) 
• EKG on day 7 
• Get the study drug 
mithramycin 
• Twice daily for 24 hours after 
the last dose of study drug 
receive oral/IV dexamethasone  
Day 
9 
to 28 
Come into the clinic and do the 
following: 
• Routine blood tests twice per week 
• Physical exam by your doctor 
every week 
Day 9 
to 28 
Come into the clinic and do the 
following: 
• Routine blood tests twice per 
week (cycles 2 and 3) 
• Routine blood tests weekly for 
cycles 4 to the end 
• Physical exam by your doctor 
every week 
 
Every 
2 
cycles 
Come into the clinic and do the 
following: 
• Routine blood tests 
• Physical exam by your doctor 
• MRI or CT scan 
• Disease evaluation that will be 
done by your doctor. 
Depending on the results of 
this evaluation, your doctor 
will tell you whether or not you 
may continue the next cycle 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 9 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Birth Control 
Reproductive risks:  You should not become pregnant or father a baby while on this study 
because the drugs in this study can affect an unborn baby.  If you become pregnant on the study, 
you will be taken off of mithramycin immediately.  Further, if the pregnancy is taken to term, the 
outcome will also be recorded in study records.  Women should not breastfeed a baby while on 
this study.  It is important you understand that you need to use birth control while on this study 
and for at least two months after finishing treatment with mithramycin.  Check with your study 
doctor about what kind of birth control methods to use and how long to use them.  Some 
methods might not be approved for use in this study. 
Other medications 
There are several medications, such as aspirin or ibuprofen, which are not allowed during 
participation in this study, as these medications may increase the risk for mithramycin side 
effects, such as bleeding. It is very important that you contact your doctor prior to starting a new 
medicine (including herbal or over the counter medications) to check if it is OK to take it.  The 
study team will provide you and your home doctor with a list of medicines, which you should not 
take.  
Alternative Approaches or Treatments 
What other choices do I have if I do not take part in this study?  
 
Instead of being in this study, you have these options: 
• Getting treatment or care for your cancer without being in a study 
• Taking part in another study 
• Getting comfort care, also called palliative care.  This type of care helps reduce pain, 
tiredness, appetite problems and other problems caused by the cancer.  It does not treat 
the cancer directly.  Instead, it tries to improve how you feel.  Comfort care tries to keep 
you as active and comfortable as possible. 
Please talk to your doctor about these and other options. 
Risks or Discomforts of Participation 
What side effects or risks can I expect from being in this study?  
You may have side effects while on the study.  Everyone taking part in the study will be watched 
carefully for any side effects.  However, doctors don’t know all the side effects that may happen.  
Side effects may be mild or very serious. Your health care team may give you medicines to help 
lessen side effects.  Many side effects go away soon after you stop taking the mithramycin.  In 
some cases, side effects can be serious, long lasting, or may never go away.  Rarely there is a 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 10 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
risk of death from a serious side effect.  We will tell you if we learn any new information that 
may affect your health, welfare, or decision to stay in this study.  
 
You should talk to your study doctor about any side effects that you have while taking part in the 
study.    
Side Effects from Mithramycin 
Likely 
• Decreased blood level of calcium 
• Nausea or vomiting 
• Diarrhea 
• Loss of appetite 
• Mouth sores 
• Increase blood levels of liver enzymes, which may indicate damage to the liver (which 
may be severe) 
• Increase in LDH, which is an indicator of damage to your cells. 
Less Likely 
• Bleeding including nose bleed 
• Fever 
• Drowsiness 
• Weakness 
• Tiredness 
• Pain, redness, soreness or swelling of the area where the IV goes in the skin 
• Skin changes, blushing of the face   
• Headache 
• Mental depression, restlessness, irritability 
• Decreased platelets (the blood cell that helps blood clot) 
• Decreased red blood cells (the blood cells that carry oxygen) 
• Abnormal levels of white blood cells (the cells that prevent infection) 
• Muscle or stomach cramps, possibly due to a low blood level of calcium  
• Abnormal levels of blood elements including phosphorus or potassium. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 11 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
• Increase blood levels of liver enzymes, which may indicate damage to the liver 
• Increased blood levels of kidney function tests, such as creatinine, urea or protein 
Rare but Serious 
• Bleeding that may require a transfusion of blood or blood products.  Bleeding may result 
in bloody or black stools or vomiting blood, or blood nose, or small broken blood vessels 
under the skin 
• A significant rash called toxic epidermal necrolysis characterized by widespread and 
severe skin irritation has been reported with mithramycin. 
While receiving mithramycin, you should avoid any drugs that contain salicylates, a component 
of aspirin, such as Aspirin, Bufferin, Ascriptin, Aspergum, Anacin, and some Alka-Seltzer 
products and some cold preparations, as they may increase the risk of bleeding. 
When the WBC is low (neutropenia), fever may be a sign of significant infection. When fever 
and neutropenia is present, hospitalization for administration of intravenous antibiotics may be 
necessary. 
Because mithramycin may decrease the production of red cells (the part of the blood that carries 
oxygen and gives energy) and platelets (the part of the blood that helps prevent bleeding), it is 
possible that you will require transfusions with red cells or platelets. When these transfusions are 
required will vary from participant to participant and can be discussed with your doctor. Most 
transfusions have no side effects.  However, you may have an allergic reaction (a rash, hives or 
very rarely, difficulty breathing that can be so severe as to result in death).  In addition, although 
the blood bank that services our patients is excellent, there is at least a theoretic possibility of 
infections such as AIDS or hepatitis being transmitted by blood.  The blood bank is very good at 
screening for these diseases and the chance of these problems is very small.  A separate informed 
consent would be obtained should you need a blood transfusion. 
 
Side Effects from Dexamethasone 
• 
High blood pressure 
• 
High Blood glucose 
• 
Stomach upset 
• 
Burning in stomach from excess stomach acid 
• 
Increased risk of infection 
• 
Increased appetite  
• 
Weight gain 
• 
Thinning of bones  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 12 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
• 
Increase pressure in eyes 
• 
Personality changes, e.g., irritability, euphoria, mania 
• 
Feelings of depression 
• 
Pulmonary tuberculosis 
• 
Vision problems 
• 
Acne, allergic dermatitis, dry scaly skin 
• 
Destruction of bone tissue 
Potential Benefits of Participation 
Are there benefits to taking part in this study?  
The potential benefit of the treatment with mithramycin is that it may cause your cancer to stop 
growing or to shrink for a period of time.  It may lessen the symptoms, such as pain, that are 
caused by the cancer.  It is extremely unlikely that this treatment will cure your cancer.  Because 
there is not much information about the mithramycin effect on cancers in humans, we do not 
know if you will benefit from taking part in this study.  Information learned from this study may 
help future patients with cancer. 
Research Subject’s Rights 
What are the costs of taking part in this study?  
If you choose to take part in the study, the following will apply, in keeping with the NIH policy: 
• 
You will receive study treatment at no charge to you. This may include 
surgery, medicines, laboratory testing, x-rays or scans done at the 
Clinical Center, National Institutes of Health (NIH), or arranged for you 
by the research team to be done outside the Clinical Center, NIH if the 
study related treatment is not available at the NIH.  
• 
There are limited funds available to cover the cost of some tests and 
procedures performed outside the Clinical Center, NIH.  You may have 
to pay for these costs even if they are not covered by your insurance 
company. 
• 
Medicines that are not part of the study treatment will not be provided or 
paid for by the Clinical Center, NIH. 
• 
Once you have completed taking part in the study, medical care will no 
longer be provided by the Clinical Center, NIH. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 13 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Will my medical information be kept private?  
We will do our best to make sure that the personal information in your medical record will be 
kept private.  However, we cannot guarantee total privacy.  Your personal information may be 
given out if required by law.  If information from this study is published or presented at scientific 
meetings, your name and other personal information will not be used.  
Organizations that may look at and/or copy your medical records for research, quality assurance, 
and data analysis include: 
• Representatives of the  National Cancer Institute (NCI) and other government agencies, 
like the Food and Drug Administration (FDA), involved in overseeing research 
• The Institutional Review Board (IRB) of this hospital 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law.  This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results.  You can search this Web site at any time.” 
Stopping Therapy  
You can stop taking part in the study at any time.  However, if you decide to stop taking part in 
the study, we would like you to talk to the study doctor and your regular doctor first. 
 
Conflict of Interest 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of 
interest. 
 
The 
following 
link 
contains 
details 
on 
this 
process 
http://ethics.od.nih.gov/procedures/COI-Protocol-Review-Guide.pdf.  You may ask your 
research team for a copy of the Protocol Review Guide or for more information.  Members of the 
research team who do not work for NIH are expected to follow these guidelines but they do not 
need to report their personal finances to the NIH. 
 
Members of the research team working on this study may have up to $15,000 of stock in the 
companies that make products used in this study.  This is allowed under federal rules and is not a 
conflict of interest.  
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 14 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Optional Studies  
We would like to keep some of the specimens and data that are collected for future research.  
These specimens and data will be identified by a number and not your name.  The use of your 
specimens and data will be for research purposes only and will not benefit you.  It is also 
possible that the stored specimens and/or data may never be used.  Results of research done on 
your specimens and data will not be available to you or your doctor.  It might help people who 
have cancer and other diseases in the future.   
 
If you decide now that your specimens and data can be kept for research, you can change your 
mind at any time. Just contact us and let us know that you do not want us to use your specimens 
and/or data.  Then any specimens that remain will be destroyed, and your data will not be used 
for future research.  
Please read each sentence below and think about your choice. After reading each sentence, circle 
and initial the answer that is right for you.  No matter what you decide to do, it will not affect 
your care. 
1. My specimens and data may be kept for use in research to learn about, prevent, or treat cancer.  
 
Yes              No               Initials_________ 
 
2. My specimens and data may be kept for use in research to learn about, prevent or treat other 
health problems (for example: diabetes, Alzheimer's disease, or heart disease).  
 
Yes              No               Initials_________ 
 
3. Someone may contact me in the future to ask permission to use my specimens and/or data in 
new research not included in this consent.  
 
Yes              No               Initials_________ 
 
 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 
12-C-0135 
CONTINUATION: page 15 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
(Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
OTHER PERTINENT INFORMATION 
1. 
Confidentiality.  When results of an NIH research study are reported in medical journals 
or at scientific meetings, the people who take part are not named and identified.  In most cases, 
the NIH will not release any information about your research involvement without your written 
permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This 
information might affect (either favorably or unfavorably) the willingness of the insurance 
company to sell you insurance. 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you 
should know that the Act allows release of some information from your medical record without 
your permission, for example, if it is required by the Food and Drug Administration (FDA), 
members of Congress, law enforcement officials, or authorized hospital accreditation 
organizations. 
2. 
Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-
term medical care for any injury resulting from your participation in research here.  In general, 
no long-term medical care or financial compensation for research-related injuries will be 
provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  
However, you have the right to pursue legal remedy if you believe that your injury justifies such 
action. 
3. 
Payments.  The amount of payment to research volunteers is guided by the National 
Institutes of Health policies.  In general, patients are not paid for taking part in research studies at 
the National Institutes of Health.  Reimbursement of travel and subsistence will be offered 
consistent with NIH guidelines. 
4. 
Problems or Questions.  If you have any problems or questions about this study, or 
about your rights as a research participant, or about any research-related injury, contact the 
Principal Investigator, Dr. Brigitte Widemann at Telephone: 301-496-7387, or Ms. Donna 
Bernstein, Building 10, Room 13C101, research nurse for the study, Telephone: 301-435-7804. 
You may also call the Clinical Center Patient Representative at (301) 496-2626. If you have any 
questions about the use of your specimens and data for future research studies, you may also 
contact the Office of the Clinical Director, Telephone:  301-496-4251. 
5. Consent Document.  Please keep a copy of this document in case you want to read it again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER:  12-C-0135 
CONTINUATION: page 16 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
(Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
COMPLETE APPROPRIATE ITEM(S) BELOW: 
A. 
Adult Patient’s Consent B. 
Parent’s Permission for Minor Patient. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby consent to 
take part in this study. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby give 
permission for my child to take part in this 
study.   
(Attach NIH 2514-2, Minor’s Assent, if 
applicable.) 
 
 
 
 
Signature of Adult Patient/ 
Legal Representative 
Date 
Signature of Parent(s)/ Guardian Date 
 
Print Name 
 
Print Name 
C.  Child’s Verbal Assent (If Applicable) 
The information in the above consent was described to my child and my child agrees to 
participate in the study. 
 
 
 
Signature of Parent(s)/Guardian 
Date 
Print Name 
THIS CONSENT DOCUMENT HAS BEEN APPROVED FOR USE  
FROM JANUARY 12, 2015 THROUGH JANUARY 11, 2016. 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
Print Name 
 
Print Name 
 

Prot_SAP_002.pdf: 
Amendment
Protocol Number:
12C0135-F
Principal Investigator: Brigitte Widemann NCI 
POB 
301.496.7387 
widemanb@mail.nih.gov
(NIH Employee Name, Institute/Branch, Telephone and e-mail)
Reference Number:
364514
Protocol Title:
Phase I/II Trial of Mithramycin in Children and Adults with Refractory Extracranial Solid Tumors (Phase I) or Ewing Sarcoma and EWS-
FLI1 Fusion Transcript (Phase II)
SIGNATURES
Principal Investigator (*): 
Brigitte Widemann - applied signature on 12/06/2016 11:51 AM EST
Accountable Investigator:
PI is the Accountable Investigator
Branch Chief/CC Department Head (**):
Brigitte Widemann - applied signature on 12/06/2016 11:51 AM EST
Medical Advisory Investigator (if applicable):
N/A
Lead Associate Investigator signature:
N/A
Referral Contact signatures:
N/A
Associate Investigators signatures:
N/A
For Institute/Center Scientific Review Committee:
N/A
Other IC Clinical Director signatures:
N/A
APPROVALS
IRB Chair:
Michael Hamilton - applied signature on 12/16/2016 5:44 PM EST
Clinical Director:
N/A
CONCURRENCE
OPS Protocol Specialist:
Signature
Print Name
Date
* Signature signifies that investigators on this protocol have been informed that the collection and use of personally identifiable information at
the NIH are maintained in a system of record governed under provisions of the Privacy Act of 1974. The information provided is mandatory for
employees of the NIH to perform their assigned duties as related to the administration and reporting of intramural research protocols and
used solely for those purposes.  Questions may be addressed to the Protrak System Owner.
** I have reviewed this research project and considered the NIH Policy for Inclusion of Women and Minorities in Clinical Research. Taking into 
account the overall impact that the project could have on the research field involved, I feel the current plans adequately includes both sex/
gender, minorities, children, and special populations, as appropriate. The current enrollment is in line with the planned enrollment report for 
inclusion of individuals on the basis of their sex/gender, race, and ethnicity and is appropriate and of scientific and technical merit. 
IRB Meeting Date:
Expedited
DEC Clearance Date:
11/21/2016
Protocol Version Date:11/09/2016
Lemuel Clayborn                                                                                  AM F                                              12/21/2016


Abbreviated Title: Ph I/II Pediatric Mithramycin 
Version Date:  11/09/2016 
 
Confidential 
 
1 
Abbreviated Title:  Ph I/II Pediatric Mithramycin 
NCI Protocol #: 12C0135 F 
Version Date:  11/09/2016 
Title:  Phase I/II Trial of Mithramycin in Children and Adults with Refractory Extracranial Solid 
Tumors (Phase I) or Ewing Sarcoma and EWS-FLI1 Fusion Transcript (Phase II) 
Principal Investigator:    
 
 
Brigitte Widemann, M.D.A-E 
 
 
Pediatric Oncology Branch, CCR, NCI 
 
 
10 Center Drive CRC, Room 1-5750 
 
 
Bethesda, MD 20892-1101 
 
 
Phone: (240) 760-6203 
 
 
E-mail: widemanb@mail.nih.gov 
NIH Associate Investigators:    
Fernanda Arnaldez, M.D, POB, CCR, NCIA-E 
 
*Paul Jarosinski, PharmD, Pharmacy, CC, NIHA,B 
Joanne Derdak, NP, POB, CCR, NCIA-E 
Jay Lozier, M.D., DLM, CC, NIHA,B 
*Choh Yeung, POB, CCR, NCIE,F 
*Eva Dombi, M.D., POB, CCR, NCIE,F 
 
*Seth M. Steinberg, Ph.D., OCD, CCR, NCIE,F 
*Min He, Ph.D., DTP, DCTD, NCIE,F 
* Mark Raffeld, M.D., LP, CCR, NCIE,F 
 
John Glod, M.D., Ph.D., POB, CCR, NCIA-E 
Amanda Carbonell, R.N., OCD, CCR, NCIA,B,E 
Donna Bernstein, R.N., OCD, CCR, NCIA,B,E  
*Not responsible for direct patient care 
Referral Contact/ 
Study Coordinator:   
 
Donna Bernstein, R.N., OCD, CCR, NCI 
 
 
10 Center Drive CRC, Room 1-3750 
 
 
Bethesda, MD 20892-1106 
 
 
Phone: (240) 760-6189 
 
 
 
 
 
E-mail: bernsted@mail.nih.gov 
Investigator Roles 
A. Obtain information by intervening or interacting with 
living individuals for research purposes 
B. Obtaining identifiable private information about 
living individuals 
C. Obtaining the voluntary informed consent of 
individuals to be subjects 
D. Makes decisions about subject eligibility 
E. Studying, interpreting, or analyzing identifiable 
private information or data/specimens for research 
purposes 


Abbreviated Title: Ph I/II Pediatric Mithramycin 
Version Date:  11/09/2016 
 
Confidential 
 
2 
F. Studying, interpreting, or analyzing de-identified data 
or specimens for research purposes 
 
Investigational Agent:     
Drug Name: 
Mithramycin 
IND Number: 
115272 
Sponsor: 
Center for Cancer Research, NCI 
 
 
 


Abbreviated Title: Ph I/II Pediatric Mithramycin 
Version Date:  11/09/2016 
 
Confidential 
 
3 
PRÉCIS  
Background: 
• Mithramycin, an anti-tumor antibiotic, underwent broad clinical evaluation in solid tumors 
and leukemias in the 1960’s and demonstrated activity in some leukemias, lymphomas, 
and solid tumors. In particular, mithramycin was found to have activity against testicular 
cancers and was briefly used in the clinic for this tumor prior to the development of the 
currently used treatment regimen. 
• The Ewing Sarcoma Family of Tumors (ESFT) is the second most common malignant bone 
tumor of childhood. There has been very little improvement in overall patient survival in 
past years, particularly for patients with high risk metastatic or relapsed disease.  Therefore, 
there is a need for effective novel agents for the treatment of this disease. 
• Multiple studies have shown that suppressing the expression of EWS-FLI1 effectively 
limits the tumorigenicity of ESFT cells.  Laboratory studies have shown that mithramycin 
effectively suppresses the activity of EWS-FLI1 both in vitro and in vivo.  
Objectives (primary): 
• Phase I portion of this study is to: determine the tolerability, toxicity, and the 
recommended phase II dose of mithramycin in children and adolescents with refractory 
extracranial solid tumors.  
• Phase II portion of this trial is to: determine the objective response rate (CR and PR) of 
Ewing sarcoma to mithramycin in children and adults using RECIST criteria when 
administered at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated 
every 28 days until unacceptable toxicity or disease progression. 
• Phase II portion of this trial to: evaluate if mithramycin inhibits NR0B1 in tumor tissue and 
determine changes in gene expression signature pre-treatment and at steady state on day 
+4 of treatment in patients ≥ 18 years old with Ewing sarcoma and EWS/FLI1 fusion 
transcript with disease amenable to percutaneous biopsy. 
Eligibility: 
• Phase I Portion:  children (≥ 12 months) and adolescents (≤ 17 years) with recurrent or 
refractory extracranial solid tumors. 
• Phase II Portion in adults:  adults (≥ 18 years of age at enrollment) with recurrent or 
refractory measurable extracranial Ewing sarcoma and the EWS-FLI1 fusion transcript. 
• Phase II Portion in children and adolescents: Once the adult dose is deemed safe, children 
(≥ 12 months) and adolescents (≤ 17 years) with recurrent or refractory measurable 
extracranial Ewing sarcoma and the EWS-FLI1 fusion transcript will begin enrollment to 
the Phase II portion. 
• Participants must meet safety laboratory criteria and prior therapy limitations. 
Design: 
• Phase I Portion: Mithramycin will be administered in escalating doses to children and 
adolescents intravenously over 6 hours once daily for 7 days to be repeated every 28 days 
until unacceptable toxicity or disease progression.  Mithramycin will be given with 


Abbreviated Title: Ph I/II Pediatric Mithramycin 
Version Date:  11/09/2016 
 
Confidential 
 
4 
dexamethasone prophylaxis to ameliorate hepatotoxicity.  The cohort at the recommended 
dose or MTD will be expanded up to 12 patients, and attempts will be made to enroll 6 
patients that are ≥12 years of age and 6 patients that are <12 years of age to gain experience 
with a broad age range of patients. A maximum of 18 evaluable patients will be enrolled 
on the phase I portion.  
• Phase II Portion:  Using a Simon two stage design, mithramycin will be administered 
intravenously at 17.5 microgram/kg over 6 hours once daily for 7 days to be repeated 
every 28 days until unacceptable toxicity or disease progression to children and adults 
with Ewing sarcoma with EWS-FLI1 fusion transcript.  Mithramycin will be given with 
dexamethasone prophylaxis to ameliorate hepatotoxicity.  Up to 24 evaluable patients 
will be enrolled on the phase II portion. 
• The Phase I and Phase II portions of the protocol will enroll patients simultaneously. 
 
 


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TABLE OF CONTENTS  
Précis ..........................................................................................................................................3 
TABLE OF CONTENTS ............................................................................................................5 
1 
INTRODUCTION ............................................................................................................. 10 
1.1 
Study Objectives ......................................................................................................... 10 
1.1.1 
Primary Objectives............................................................................................... 10 
1.1.2 
Secondary Objectives ........................................................................................... 10 
1.2 
Background and Rationale .......................................................................................... 10 
1.2.1 
Ewing Sarcoma Family of Tumors (ESFT) .......................................................... 10 
1.2.2 
Preclinical studies ................................................................................................ 12 
1.2.3 
Clinical studies in adults ...................................................................................... 13 
1.2.4 
Clinical activity .................................................................................................... 14 
1.2.5 
Clinical studies in children ................................................................................... 15 
1.2.6 
Clinical Protocol 12C0135 ................................................................................... 15 
1.3 
Rationale summary ..................................................................................................... 15 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ..................................................... 16 
2.1 
Eligibility Criteria ....................................................................................................... 16 
2.1.1 
Inclusion Criteria ................................................................................................. 16 
2.1.2 
Exclusion Criteria ................................................................................................ 18 
2.1.3 
Recruitment Strategies ......................................................................................... 19 
2.2 
Screening Evaluation .................................................................................................. 19 
2.2.1 
General ................................................................................................................ 19 
2.2.2 
Laboratory ........................................................................................................... 19 
2.2.3 
Radiographic Evaluation ...................................................................................... 20 
2.2.4 
Cardiac Function Studies ..................................................................................... 20 
2.2.5 
Pathologic/Tissue Evaluation ............................................................................... 20 
2.3 
Registration Procedures............................................................................................... 20 
2.3.1 
Registration at NCI .............................................................................................. 21 
3 
STUDY IMPLEMENTATION .......................................................................................... 21 
3.1 
Study Design............................................................................................................... 21 
3.1.1 
Dose Limiting Toxicity ........................................................................................ 22 
3.1.2 
Dose Escalation in Phase I Portion ....................................................................... 23 


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3.1.3 
Phase II Portion.................................................................................................... 23 
3.2 
Drug Administration ................................................................................................... 24 
3.3 
Dose Reduction or Modifications ................................................................................ 24 
3.4 
Criteria for Additional Cycles ..................................................................................... 25 
3.5 
On Study Protocol Evaluation (Appendix 3: Study Calendar) ...................................... 25 
3.5.1 
Monitoring During Active Treatment: .................................................................. 25 
3.5.2 
Monitoring after completion of treatment with mithramycin: ............................... 26 
3.5.3 
Off Study Evaluation (at least 30 days after last dose) .......................................... 26 
3.6 
Concurrent Therapies .................................................................................................. 26 
3.7 
Criteria for Removal from Protocol Therapy and Off Study Criteria ............................ 26 
3.7.1 
Criteria for removal from protocol therapy ........................................................... 26 
3.7.2 
Criteria for removal from Study ........................................................................... 27 
3.7.3 
Off Study Procedure ............................................................................................ 27 
4 
CONCOMITANT MEDICATIONS AND SUPPORTIVE MEASURES ........................... 27 
4.1 
Management of Bleeding, Hematologic and Blood Product Support ............................ 27 
4.2 
Antiemetic Therapy..................................................................................................... 28 
4.3 
Hepatotoxicity Prophylaxis using dexamethasone ....................................................... 28 
4.4 
Antidiarrheals ............................................................................................................. 28 
4.5 
Electrolyte Replacement ............................................................................................. 28 
4.6 
QTc Prolongation ........................................................................................................ 28 
5 
Correlative studies / Biospecimen Collection ..................................................................... 28 
5.1 
Pharmacokinetic (PK) Sampling ................................................................................. 28 
5.2 
Studies on Tumor tissue .............................................................................................. 29 
5.2.1 
Evaluation of NR0B1 expression on archival tumor tissue ................................... 29 
5.2.2 
Confirmation of EWS/FLI1 fusion transcript for eligibility for phase II portion: .. 29 
5.2.3 
Evaluation of NR0B1 expression from mandatory tumor biopsies ........................ 29 
5.2.4 
Other Investigations (See Appendix 7: Studies to be performed on tumor tissue)
 
29 
5.2.5 
Future Research Studies ....................................................................................... 30 
5.3 
Sample Storage, Tracking and Disposition .................................................................. 30 
5.3.1 
Pediatric Research Samples: Blood Drawing Limits for Research Purposes ......... 30 
5.3.2 
NIH Policy on Research Use of Stored Human Samples, Specimens, or Data ...... 30 
6 
DATA COLLECTION AND EVALUATION ................................................................... 31 


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6.1 
Data Collection ........................................................................................................... 31 
6.1.1 
Source Documents ............................................................................................... 31 
6.2 
Response Criteria ........................................................................................................ 31 
6.2.1 
Response Criteria for Radiographic Studies ......................................................... 31 
6.2.2 
Confirmatory Measurement/Duration of Response ............................................... 33 
6.2.3 
Measurable Disease ............................................................................................. 34 
6.2.4 
Malignant Lymph Nodes ...................................................................................... 34 
6.2.5 
Non-Measurable Disease...................................................................................... 34 
6.2.6 
Target Lesions ..................................................................................................... 34 
6.2.7 
Non-Target Lesions ............................................................................................. 35 
6.2.8 
Metastatic Bone Lesions ...................................................................................... 35 
6.2.9 
Guidelines for Evaluation of Measurable Disease ................................................. 35 
6.2.10 Methods of Measurement ..................................................................................... 35 
6.2.11 Additional response evaluation using volumetric analysis .................................... 36 
6.3 
Toxicity Criteria .......................................................................................................... 36 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
 
36 
7.1 
Definitions .................................................................................................................. 36 
7.1.1 
Adverse Event...................................................................................................... 36 
7.1.2 
Suspected adverse reaction ................................................................................... 36 
7.1.3 
Unexpected adverse reaction ................................................................................ 37 
7.1.4 
Serious ................................................................................................................. 37 
7.1.5 
Serious Adverse Event (SAE) .............................................................................. 37 
7.1.6 
Disability ............................................................................................................. 37 
7.1.7 
Life-threatening adverse drug experience ............................................................. 37 
7.1.8 
Protocol Deviation (NIH Definition) .................................................................... 37 
7.1.9 
Non-Compliance (NIH Definition) ....................................................................... 38 
7.1.10 Unanticipated Problem ......................................................................................... 38 
7.2 
NCI-IRB and NCI Clinical Director Reporting ............................................................ 38 
7.2.1 
NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths
 
38 
7.2.2 
NCI-IRB Requirements for PI Reporting at Continuing Review ........................... 38 
7.2.3 
NCI-IRB Reporting of IND Safety Reports .......................................................... 39 


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7.2.4 
Request for waiver from IRB reporting: Anticipated PD and Expedited Non-UP 
AEs 
39 
7.3 
IND Sponsor Reporting Criteria .................................................................................. 39 
7.3.1 
Expedited Adverse Event Reporting Criteria to the IND Sponsor ......................... 39 
7.3.2 
Annual Reporting of AEs to the Sponsor .............................................................. 39 
7.4 
FDA Reporting Criteria ............................................................................................... 39 
7.4.1 
IND Safety Reports to the FDA (Refer to 21 CFR 312.32) ................................... 39 
7.4.2 
FDA Annual Reports (Refer to 21 CFR 312.33) ................................................... 40 
7.5 
Data and Safety Monitoring Plan................................................................................. 40 
7.5.1 
Principal Investigator/Research Team .................................................................. 40 
7.5.2 
Sponsor Monitoring Plan ..................................................................................... 40 
8 
STATISTICAL CONSIDERATIONS ............................................................................... 40 
8.1 
Phase I Portion ............................................................................................................ 40 
8.2 
Phase II Portion ........................................................................................................... 41 
8.2.1 
Methods of Analysis ............................................................................................ 41 
8.3 
Evaluation of Response ............................................................................................... 42 
8.4 
Inhibition of NR0B1 in Tumor Tissue and Changes in Gene Expression Signature ..... 42 
8.5 
Secondary Endpoints ................................................................................................... 42 
8.6 
Number and Rate of Accrual ....................................................................................... 43 
9 
HUMAN SUBJECTS PROTECTIONS ............................................................................. 43 
9.1 
Rationale For Subject Selection ................................................................................... 43 
9.2 
Participation of Children ............................................................................................. 43 
9.3 
Evaluation of Benefits and Risks/Discomforts ............................................................. 43 
9.4 
Consent and Assent Process and Documentation ......................................................... 44 
10 PHARMACEUTICAL INFORMATION .......................................................................... 44 
10.1 
Mithramycin (Plicamycin) ....................................................................................... 44 
10.1.1 Source .................................................................................................................. 44 
10.1.2 Toxicity ............................................................................................................... 44 
10.1.3 Formulation and preparation ................................................................................ 45 
10.1.4 Stability and Storage ............................................................................................ 46 
10.1.5 Administration procedures ................................................................................... 46 
10.1.6 Contraindications ................................................................................................. 46 
10.2 
DEXAMETHASONE ................................................................................................... 47 


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10.2.1 Source:................................................................................................................. 47 
10.2.2 Toxicity: .............................................................................................................. 47 
10.2.3 Formulation and preparation: ............................................................................... 47 
10.2.4 Administration procedures: .................................................................................. 47 
10.2.5 Incompatibilities .................................................................................................. 47 
11 APPENDICES .................................................................................................................. 48 
11.1 
Appendix 1: Mithramycin Clinical Experience Reported in the Literature................ 48 
11.2 
Appendix 2:  Performance Status Scales/Scores ....................................................... 52 
11.3 
Appendix 3: Study Calendar .................................................................................... 53 
11.4 
Appendix 4: Pharmacokinetic Worksheet for Mithramycin (Phase I and Phase II) For 
nursing staff .......................................................................................................................... 56 
11.5 
Appendix 5: Pharmacokinetic Worksheet for Mithramycin (Phase I and Phase II) For 
Laboratory Personnel and Research Nurses ........................................................................... 59 
11.6 
Appendix 6: Serum Sample For Potential Biomarker (Phase I and Phase II) ............ 62 
11.7 
Appendix 7: Studies to be performed on tumor tissue .............................................. 63 
11.8 
Appendix 8: Plasma samples for clinical purpose .................................................... 65 
11.9 
Appendix 9: Imaging studies for volumetric analysis of tumor burden ..................... 66 
11.10 
Appendix 10: Patient Log for Reactions/Symptoms ................................................. 67 
11.11 
Appendix 11: Patient Log for Concomitant Medication ........................................... 68 
11.12 
Appendix 12:  Monitoring Toxicities Between Mithramycin Cycles with Local 
Physician ............................................................................................................................... 69 
12 References ......................................................................................................................... 70 
 
 
 


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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objectives 
1.1.1.1 Phase I portion of this study is to: determine the tolerability, toxicity, and recommended 
dose of mithramycin in children and adolescents with refractory extracranial solid 
tumors.  
1.1.1.2 Phase II portion of this trial is to: determine the objective response rate (CR and PR) of 
Ewing sarcoma with EWS-FLI1 fusion transcript to mithramycin in children and adults 
using RECIST criteria when administered at 17.5 microgram/kg over 6 hours once daily 
for 7 days to be repeated every 28 days until unacceptable toxicity or disease 
progression.  If the pediatric phase II dose of mithramycin differs from the adult phase 
II dose, the protocol will be amended to address accrual and response rate to 
mithramycin in children in a separate stratum. 
1.1.1.3 Evaluate if mithramycin inhibits NR0B1 in tumor tissue, and determine changes in 
EWS/FLI1 gene expression signature by obtaining mandatory tumor biopsies pre 
treatment and at steady state on day +4 of treatment in patients ≥ 18 years old with 
Ewing sarcoma and EWS/FLI1 fusion transcript and disease amenable to percutaneous 
biopsy (phase II). 
1.1.2 Secondary Objectives 
1.1.2.1 Describe pharmacokinetic parameters for each dose level using non-compartmental 
methods (phase I and II) 
1.1.2.2 Evaluate NR0B1 expression in patients with Ewing sarcoma using 
immunohistochemistry on archival tumor tissue, if available (phase I and II) 
1.1.2.3 To prospectively evaluate the feasibility and utility of volumetric analysis of target 
lesions, and descriptively compare volumetric measurements to 1D (RECIST), and 2D 
(WHO) measurements (phase I and II). 
1.1.2.4 Define preliminarily time to progression (TTP) in patients with Ewing sarcoma (phase 
II) 
1.2 
BACKGROUND AND RATIONALE 
1.2.1 Ewing Sarcoma Family of Tumors (ESFT) 
The Ewing Sarcoma Family of Tumors (ESFT) is the second most common malignant bone tumor 
of childhood (1, 2).  In the past 25 years since the advent of 5-drug ESFT therapy, there has been 
very little improvement in overall patient survival particularly for patients with high risk metastatic 
or relapsed disease (1).  Therefore, there exists a great need for agents that target the genes this 
tumor depends on for survival. 
All ESFT tumors have fusion oncogenic transcription factors generated by chromosomal 
translocations involving the EWS gene and one of the ETS (E26 transformation-specific) family 
of transcription factors (3, 4)  These transcription factors are highly dysregulated and believed to 
be critical to the biology of these tumors mediating both malignant transformation and progression 
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In 85% of cases, the fusion transcription factor is EWS-FLI1 usually generated by the 
t(11;22)(q24;q12) chromosomal translocation (4).  This transcription factor contains the binding 
domain of FLI1 and the transactivating domain of EWS but loses most of the regulatory domains 
of both proteins (6).  Therefore, this protein is constitutively active and alters the expression of 
more than 500 genes (7).  The resulting genetic program is believed to drive malignant 
transformation and clearly plays a central role in mediating the oncogenic phenotype (8-10).  Most 
notably, EWS-FLI1 has been shown to directly or indirectly mediate the evasion of apoptosis and 
senescence, drive proliferation and even participate in the regulation of genes involved in 
metastasis (11-15).  In addition, multiple groups have shown that knockdown of EWS-FLI1 with 
antisense DNA, siRNA or dominant negative methods is incompatible with ESFT cellular survival 
(16).  Therefore, recent preclinical efforts have been focused on developing a small molecule 
therapy targeting this transcription factor (17, 18).  Unfortunately, previously identified small 
molecule inhibitors have failed to demonstrate clinical activity against ESFT (19). 
We have recently completed a high throughput screen of more than 50,000 compounds to identify 
mithramycin as an inhibitor of EWS-FLI1.  We have utilized a novel approach combining a 
luciferase based primary screen and a gene signature multiplex PCR secondary screen to identify 
compounds that block the activity of EWS-FLI1 (20). In the primary screen, we required 
compounds to suppress the expression of luciferase driven by the promoter of a well- characterized 
EWS-FLI1 downstream target, NR0B1 without changing the expression of a constitutively active 
CMV driven promoter or decreasing cell viability as measured by an XTT assay (Figure 1).  By 
performing these three screens in parallel, we were able to control for non-specific cytotoxic agents 
and general transcription inhibitors. We subsequently required the compounds to reverse a gene 
signature of more than 10 downstream targets of EWS-FLI1 utilizing a novel multiplex PCR assay 
that we developed to sort and prioritize compounds for further validation.   
 
The compound that most effectively suppressed 
EWS-FLI1 activity was mithramycin (20).  We 
showed that mithramycin suppresses the mRNA 
expression of our gene signature of EWS-FLI1 
targets as well as an independent published gene 
signature of 50 different EWS-FLI1 targets.  In 
addition, 
the 
suppression 
of 
EWS-FLI1 
downstream targets also occurs at the protein 
level and leads to the induction of apoptosis in 
ESFT cells.  These effects translate into a 
significant reduction of growth of two different 
xenograft models of Ewing sarcoma with one 
xenograft virtually eliminated from the mouse 
with mithramycin treatment (20).  Finally, we 
found evidence of activity of mithramycin in 
patients with Ewing sarcoma.  Five patients with advanced, widely metastatic Ewing sarcoma were 
treated with mithramycin in the 1960s and at least one durable complete response was reported 
(21).  The findings in our screen combined with the preclinical and clinical activity of mithramycin 
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and young adults with refractory solid tumors and Ewing sarcoma and EWS-FLI1 fusion 
transcript.  
1.2.2 Preclinical studies 
Mithramycin effectively suppresses the activity of EWS-FLI1 both in vitro and in vivo.  We have 
shown by multiplex PCR, quantitative real time PCR and microarray expression profiling that 
mithramycin reverses the induction of two independently generated gene signatures of downstream 
targets of EWS-FLI1 (20).  In addition, we have demonstrated that mithramycin suppresses the 
protein expression of two well-characterized downstream targets of EWS-FLI1, ID2 and NR0B1 
in two different ESFT cell lines in vitro at nanomolar concentrations (22-24). The timing of this 
suppression correlates well with the induction of apoptosis and translates to an IC50 of 10-15 nM 
in a panel of ESFT cell lines.   
Finally, we used a xenograft model to show suppression of growth of two different ESFT 
xenografts.  The TC32 xenograft model showed a regression of tumor in the first week of treatment 
that persisted long after every mouse in the control had to be sacrificed for progression.  The TC71 
xenograft also showed an impressive suppression in growth that translated into a doubling in 
survival for the affected mice.  In contrast, despite relatively similar in vitro IC50s, an osteosarcoma 
MNNG-HOS xenograft showed minimal suppression in growth and no improvement in survival 
with mithramycin treatment.   In addition, we were able to recapitulate the biochemistry observed 
in vitro by utilizing immunohistochemistry to demonstrate suppression of NR0B1, an EWS-FLI1 
downstream target, in the mithramycin treated but not the control tumor tissue (20). 
In summary, ESFT depends on the continued expression of EWS-FLI1 for cell survival.  We have 
completed a high throughput screen to identify mithramycin as an inhibitor of EWS-FLI1 and 
shown suppression of activity both in vitro and in vivo.  As predicted, blockade of EWS-FLI1 
causes the induction of apoptosis, leads to a nM in vitro IC50 and suppresses growth of ESFT 
xenografts. 
1.2.2.1 Preclinical toxicology  
In mice, the LD50 was determined to be about 2000 mcg/kg (6000 mcg/m2) of body weight.  This 
dose is higher than the 100 mcg/kg of body weight that was found to be lethal in some dogs (2000 
mcg/m2) and monkeys (1200 mcg/m2) (25).  However, the drug was found to be essentially non-
toxic in dogs and monkeys administered at a dose of 24-50 mcg/kg/day (480-1000 mcg/m2 dog, 
288-600 µcg/m2 monkey) intravenously for 24 consecutive days (25). The most frequently 
encountered toxicities at higher doses were bleeding, anorexia, vomiting, elevated liver function 
tests, electrolyte abnormalities, bone marrow suppression and azotemia (25).  
In our experience in mice, doses of 1 mg/kg/day three times a week for 6 weeks delivered via 
intraperitoneal injection was essentially non-toxic to the mice.  At necropsy, the mice treated at 
this dose and schedule had mild hypoalbuminemia, slightly increased ALT/AST with some 
evidence of liver toxicity described as mild to moderate and minimal thrombocytopenia.   
It is notable that we were able to show that the drug interferes with the expression of the EWS-
FLI1 downstream target NR0B1 in our mouse models at this 1 mg/kg/day dose.  However, it is not 
clear if the dose of 25 mcg/kg/dose is high enough to interfere with EWS-FLI1 activity in patients.  
Therefore, one of the primary objectives will be to establish the inhibition of NR0B1 expression 
through mandatory tumor biopsies when feasible (see Section Error! Reference source not 
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well as the future clinical development of mithramycin and the preclinical development of 
mithramycin analogs.  
Administration of tritium labeled mithramycin to monkeys showed a terminal half-life of 5 hours. 
The peak cerebrospinal fluid concentration: plasma ratio was 0.12 and reached after 1 ½ hours 
(26).  Preliminary pharmacokinetic parameters of 125 I-plicamycin (mithramycin) in human plasma 
using a radioimmunoassay were described (27). Three patients were studied and received 
mithramycin at a dose ranging from 0.85 to 1.0 mg/m2 IV over 2 hours. Drug elimination was 
biphasic, with a mean elimination half-life of 10.6 hours (±1.7), and a clearance rate of 11.1± 0.4 
ml/min/m2.  
1.2.3 Clinical studies in adults 
Mithramycin underwent broad clinical evaluation in solid tumors and leukemias in the 1960’s and 
was found to have some activity against leukemias, lymphomas and carcinomas.  In particular, 
mithramycin was found to have activity against testicular cancer and was briefly used in the clinic 
for this tumor prior to the development of the currently used treatment regimen.  However, a series 
of case reports in the literature suggest a particular sensitivity of Ewing sarcoma for mithramycin 
treatment.  Prior clinical trials are summarized in Appendix 1. 
The clinical experience with mithramycin is extensive as more than 1500 patients treated with 
mithramycin are reported in the literature.  A formal Maximum Tolerated Dose (MTD) has not 
been established.  However, the earliest reports of the drug efficacy at 50 mcg/kg/dose and 
therefore established this dose daily times 5 as the optimal treatment regimen (28).  Subsequent to 
this report, a follow-up study of 58 patients found significant toxicity consisting of anorexia, 
nausea and vomiting at this dose and schedule in 70% of patients (29).  As a result, the dose of 
drug used in most studies was 25 mcg/kg/day on various schedules.  The recommended dose for 
the treatment of testicular cancer from the package insert from Pfizer was 25 mcg/kg/day daily 
times 7 (25).  In addition, it was reported that significant toxicity is dose related and occurs at 
doses above 30 mcg/kg/dose (25).  It is notable that the dose that led to the responses in the Ewing 
patients was 25 mcg/kg/day as a 8 to 24 hour infusion for 5-7 days (21).  
In general, the most frequent toxicities observed were nausea, vomiting, elevated liver function 
tests, infusional fever, mucositis, bleeding tendencies, thrombocytopenia, electrolyte 
abnormalities, proteinuria and elevated BUN/Creatinine (summarized in (25)).  Unfortunately, 
there was no systematic investigation of these toxicities and it is not clear the amount of supportive 
care these patients received.  For example, nausea and vomiting occurred quite frequently, 
however, in at least two reports, the nausea responded to treatment with phenothiazines or other 
anti-emetics (30-32). 
The most profound toxicity seen was hemorrhage.  The tendency for bleeding occurred 
infrequently and in early reports was attributed to thrombocytopenia and accounted for 3 deaths 
out of 84 patients treated (33).  Subsequent studies showed that bleeding occurred early in 
treatment at about day 4 and was dose related occurring in 5.4% of 1150 patients treated at 30 
mcg/kg/day or less (25).  This time frame is significantly shorter than what is typically seen for 
chemotherapy induced thrombocytopenia.  In addition, it appears as though the platelet count 
remained >20,000/mcL in most of the cases of hemorrhage in the literature.  Subsequent to these 
studies, it was determined that mithramycin treatment caused a decrease in coagulation factors II, 
V, VII and X (34).  In addition, subsequent studies revealed that mithramycin causes abnormal 
platelet adhesion in the setting of a normal platelet count consistent with a platelet adhesion 


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molecule deficiency (35).  It is notable, that in addition to inhibiting EWS-FLI1, mithramycin is 
known to inhibit the SP1 transcription which is known to drive the biosynthesis of coagulation 
factors as well as platelet adhesion glycoproteins. Nevertheless, in all the cases of hemorrhage 
reported in the literature, it is not clear if the patients were taking any anti-coagulation medications, 
what type of blood product support was provided, and how effective the blood product support 
was in that era of treatment.  In particular, it is not clear if patients received platelets in the setting 
of a normal platelet count as would be required for an acquired platelet adhesion defect.  
Regardless of the etiology, this side effect must be approached with extreme caution. 
Another rare but significant side effect described in the literature is the report of toxic epidermal 
necrolysis that has been associated with mithramycin treatment (36, 37). This side effect is 
reported in two separate case reports but was omitted in the package insert from Pfizer (37) and 
appears to begin with plethora and non-specific skin findings.  The incidence of this side effect is 
unclear, however due to the substantial nature of this reaction, all skin eruptions should be 
approached with significant caution. 
One report describes potentiation of the cardiotoxicity of anthracyclines with mithramycin 
treatment (38).  However, this report provides no details of the cardiotoxicity observed and refers 
to mitomycin.   No other mention of cardiotoxicity has been found in the literature, package insert 
or IND that was previously filed. 
Finally, no report or investigation of cumulative toxicity could be found in the literature.  The 
closest report is one of a long term follow-up of patients with testicular carcinoma treated with 
mithramycin (39).  In this report 10 patients with metastatic testicular carcinoma out of 62 treated 
with mithramycin were available for long term follow up at 23 years.  No long-term side effect of 
the drug is reported. 
1.2.4 Clinical activity 
In 1960, the first evaluation of more than 36 patients with a variety of tumor histologies found an 
overall response rate of just under 20% defined as tumor shrinkage, improvement in performance 
status or a leveling off of body weight (28).  Subsequent to this, mithramycin was evaluated in a 
number of reports and activity was found in a variety of histologies although patient numbers and 
standard methods of evaluating responses were limited (31, 40-42).  The tumor type that seemed 
to show a consistent response was testicular carcinoma.  In one series of 305 patients with stage 
III testicular CA, 10% of patients achieved a complete response and an additional 25% showed 
some evidence of tumor regression (25).  Many of these cases were durable responses in patients 
with widely metastatic disease.  However, the overall response rate was low and the widespread 
use of the drug in this tumor, likely was limited by the development of the successful “Einhorn 
regimen” for the treatment of this cancer. 
In addition to testicular carcinoma, mithramycin was used for the treatment of malignant 
hypercalcemia with 45% of patients responding to a single dose of mithramycin in one study (43).   
However, in the same report, the bisphosphonate pamidronate was far superior and ultimately 
replaced mithramycin in this setting.   For similar reasons, mithramycin was investigated for the 
treatment of Paget’s disease of bone where it was found to have good activity in the management 
of this disorder of osteoclasts.  In one report, 10/10 patients with Paget’s disease reported 
improvements in pain and an increase in overall activity with treatment (32). 


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The activity of mithramycin in Ewing sarcoma was impressive in some of the earliest reports of 
this drug in the clinic (33).  A total of 5 patients with ESFT have been treated with mithramycin 
and reported in the literature (21). Of those 5 patients, 2 patients experienced widespread 
regressions of metastatic disease with mithramycin treatment as a single agent.  One of the two 
patients had a durable completed response and was followed for more than 7 years (21). It is not 
clear why the drug was not further investigated in ESFT.  It is likely that the advent of 
anthracycline based therapies and the evolution of combination therapies supplanted the interest 
of the community in alternative single agent therapies. 
1.2.5 Clinical studies in children 
No formal pediatric studies of mithramycin in children have been reported.  Based on the literature 
available, only 5 patients under the age of 18 received mithramycin (40).  In this published report 
of 26 patients, 5 children (ages 10-16 years of age) with embryonal carcinoma of the testis, 
malignant teratoma testis or Ewing sarcoma received total mithramycin doses of 185-250 mg/kg 
over 24 hours.  No toxicity data was reported on these five study participants.  One subject 
experienced a mixed response after the first course, one had regression of small metastases but no 
change in larger tumors, and one subject experienced transient improvement in pain reports. It is 
notable in this report that the patient with Ewing sarcoma that achieved a complete response was 
a 22 year-old adult. 
1.2.6 Clinical Protocol 12C0135 
Two adult patients with Ewing Sarcoma have been enrolled on this trial and treated at the 
25mcg/kg dose level. Following the second dose, both patients experienced grade 4, dose-
limiting AST and ALT elevation and also non dose-limiting increased LDH, GGT, and fever.  
Bilirubin remained normal and synthetic function was minimally abnormal transiently.  Both 
patients also had a fever after the 2nd dose and very mild nausea. No abdominal pain or other 
symptoms were observed.  In both cases, LFTs recovered fairly quickly after holding drug.  
Mithramycin was held after the 2nd dose for both patients.  One of the patients was retreated at a 
lower dose of 17.5 mg/kg per protocol.  Dexamethasone prophylaxis was administered in a 
similar fashion to what is done for other anti-cancer agents, which can result in hepatotoxicity, 
such as trabectedin.  The aforementioned patient was able to receive all 7 doses of mithramycin 
with transient elevation in LFTs peaking at about 200 U/L, and recovery to grade 1 or baseline 
12 days after the last dose of mithramycin. 
Based on the data noted above, we are amending the protocol to lower the phase II dose to 17.5 
mcg/kg and the phase I starting dose to 13 mcg/kg and to add dexamethasone prophylaxis with 
the goal to ameliorate hepatotoxicity.  
1.3 
RATIONALE SUMMARY 
Our high throughput screen of more than 50,000 compounds identified mithramycin as an inhibitor 
of EWS-FLI1.  ESFT has a strong dependence on EWS-FLI1 for cellular survival and we have 
shown that mithramycin interferes with this activity in vitro and in vivo and markedly suppresses 
ESFT xenograft growth.  Responses to single agent mithramycin have also been described in 
patients with refractory ESFT, but the objective response rate of mithramycin in ESFTs has not 
been determined.  These findings provide a strong rationale for the development of mithramycin 
in patients with EWS-FLI1 positive Ewing sarcoma.  


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In addition, mithramycin demonstrated activity in other adult solid tumors, and our preclinical data 
suggest that other pediatric solid tumors might also be sensitive to mithramycin treatment.  
We therefore propose a broad phase I trial of mithramycin for children and adolescents with 
refractory solid tumors to confirm tolerability of mithramycin at the recommended dose for adults 
established in the 1960s.  There will be a limited dose escalation to the adult recommended dose. 
At the adult recommended dose (or MTD) up to six children ≤12 years of age and six children ≥12 
years of age will be enrolled to gain experience with the toxicities of mithramycin over a broad 
age range.  
Simultaneous to enrolling children in the phase I portion of this trial, adults with EWS-FLI1 
positive Ewing sarcoma will enroll on the phase II portion of the trial, which has the goal to 
determine the activity of mithramycin in EWS-FLI1 positive Ewing sarcoma.  Initiating the phase 
II study in adults while determining the optimal dose in children, is based on extensive previous 
safety and dosing information obtained from clinical trials conducted in adults and the anecdotal 
reports of activity in patients with Ewing sarcoma treated in the past with mithramycin.  The Phase 
II portion will be implemented in adults using the previously approved mithramycin dose of 25 
microgram/kg administered over six hours once daily for 7 days to be repeated every 28 days.  
Once the adult phase II dose has been determined safe in children and adolescents, children will 
also be accrued to the phase II portion. Should the pediatric phase II dose of mithramycin differ 
from the adult phase II dose, the protocol will be amended to address accrual and response rate to 
mithramycin in children in a separate stratum.  The objective response rate of mithramycin will be 
determined in EWS-FLI1 positive Ewing sarcoma using a 2-stage design and targeting an objective 
response rate (RECIST PR and CR) of 25%.   
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT  
2.1 
ELIGIBILITY CRITERIA 
2.1.1 Inclusion Criteria  
2.1.1.1 Diagnosis 
Patient’s current disease state must be one for which there is no known curative therapy or 
therapy proven to prolong survival with an acceptable quality of life. 
Phase I Portion:  Measurable or evaluable refractory or recurrent extracranial solid tumors, 
excluding brain tumors and cerebral metastases.   
Phase II Portion adults and children:  Refractory or recurrent extracranial Ewing sarcoma with 
EWS-FLI1 fusion transcript.  Patients enrolled to this cohort must have measurable disease.  
Presence of the transcript will be determined during histologic confirmation of disease with a 
CLIA approved EWS-FLI paraffin assay in the Laboratory of Pathology CCR, NCI, unless a 
pathology report documenting presence of the transcript using a CLIA approved assay is obtained 
from the referring institution. 
2.1.1.2 Histologic confirmation of disease in the Laboratory of Pathology, CCR, NCI, NIH. 
2.1.1.3 Age 
Phase I Portion:  ≥ 12 months  to ≤ 17 years 
Phase II Portion in adults initially: ≥ 18 years  


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Phase II Portion expanded in pediatrics after determination of phase II dose in children will 
include children ≥ 12 months to ≤ 17 years 
2.1.1.4 Performance Score:  Karnofsky (> 10-17 years old) or Lansky (≤ 10 years old) ≥ 50%, or 
ECOG 1 or 2 (adults) (See Appendix 2) 
2.1.1.5 Prior therapy 
≥ 2 weeks must have elapsed since local palliative XRT (small port) or other substantial BM 
radiation;  
≥ 24 weeks must have elapsed since prior TBI, craniospinal XRT, or if ≥ 50% radiation of 
pelvis;  
≥12 weeks must have elapsed since stem cell transplant or infusion without TBI and no active 
graft vs. host disease;  
≥ 3 weeks must have elapsed from last dose of myelosuppressive chemotherapy (six weeks for 
nitrosoureas);  
at least 3 half-lives must have elapsed since monoclonal antibody; 
(https://members.childrensoncologygroup.org/Disc/devtherapeutics/default.asp for listing of 
monoclonal antibody half-lives.)  
≥ 7 days must have elapsed from the last dose of biologic agents. 
≥ 7 days since the completion of therapy with a growth factor 
2.1.1.6 Recovered from acute toxicities of prior therapy to ≤ Grade 1; specifically 
a) Hematologic and Coagulation Parameters  
i. 
Peripheral ANC ≥ 1000/mcL 
ii. 
Platelets ≥ 75,000/ mcL  (transfusion independent) 
iii. 
Hemoglobin ≥ 8 g/dL (PRBC transfusions permitted) 
iv. 
Normal PT/PTT with the exception of a lupus anti-coagulant, which is 
permitted, may be corrected with Vitamin K administration or transfusion.  
Fibrinogen ≥ the lower limit of normal. 
b) Hepatic Function 
i. 
Bilirubin (total) ≤ 1.5 X upper limit of normal (ULN) 
ii. 
ALT (SGPT) ≤ 2.5 X ULN 
iii. 
Albumin > 2 g/dL 
c) Renal Function 
i. 
Creatinine clearance ≥ 60 mL/min/1.73 m2, or serum creatinine base on age and 
gender as follows: 
Age (years) 
Maximum Serum Creatinine (mg/dL) 
2 to < 6 
0.8 
0.8 
6 to < 10 
1 
1 


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10 to < 13 
1.2 
1.2 
13 to < 16 
1.5 
1.4 
≥ 16 
1.7 
1.4 
• Normal calcium, magnesium and phosphorus (can be on oral 
supplementation 
2.1.1.7 Cardiac Function:   Left ventricular ejection fraction (EF) within normal institutional 
limits by Echocardiogram  MUGA or cardiac MRI. 
2.1.1.8 Ability to give informed consent. For patients <18 years of age their legal guardian 
must give informed consent.  Pediatric patients will be included in age-appropriate 
discussion in order to obtain verbal assent. 
2.1.1.9 Female and male patients (and when relevant their partners) must be willing to practice 
birth control (including abstinence) during and for two months after treatment, if of 
childbearing potential during sexual contact with a female of childbearing potential. 
2.1.1.10 A durable power of attorney (DPA) will be offered to all patients ≥ 18 years old. 
2.1.1.11 Eligibility criteria for mandatory serial tumor biopsies 
• Age: ≥ 18 years old 
• Ewing sarcoma with EWS-FLI1 fusion transcript 
• Hematologic and coagulation parameters within 2 days prior to each 
biopsy: Normal PT/PTT with exception of lupus anticoagulant, platelets 
≥75,000/mcL, peripheral ANC ≥750/mcL 
• Willing to undergo biopsies, which will only be performed on tumors 
amenable to percutaneous biopsy (Error! Reference source not found.) 
 
2.1.2 Exclusion Criteria 
2.1.2.1 Clinically significant systemic illness (e.g. serious active infections or significant 
cardiac, pulmonary, hepatic or other organ dysfunction), that in the judgment of the PI 
would compromise the patient’s ability to tolerate protocol therapy or significantly 
increase the risk of complications. 
2.1.2.2 Patients with a history intracranial Ewing sarcoma including cerebral metastases 
2.1.2.3 Patients with evidence of active bleeding, intratumoral hemorrhage or history of 
bleeding diatheses  
2.1.2.4 Patients who are receiving anticoagulants other than prophylactic anticoagulation of 
venous or arterial access devices, provided that requirements for PT, PTT and 
fibrinogen are met, as described in Section Error! Reference source not found.. 
2.1.2.5 Investigational Drugs:  Patients who are currently receiving another investigational drug 
2.1.2.6 Patients who are concurrently receiving agents, which may increase the risk for 
mithramycin related toxicities, such as hemorrhage including: 
• Thrombolytic agents  


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• Anti-inflammatory drugs, nonsteroidal (NSAIDs) or aspirin or salicylate-
containing products, which may increase risk of hemorrhage 
• Dextran  
• Dipyridamole  
• Sulfinpyrazone 
• Valproic acid     
2.1.2.7 Anti-cancer Agents:  Patients who are currently receiving other anti-cancer agents 
2.1.2.8 Lactating or pregnant females (due to risk to fetus or newborn, and lack of testing for 
excretion in breast milk). 
2.1.2.9 Patients with history of HIV, HBV or HCV due to potentially increased risk of 
mithramycin toxicity in this population.  
2.1.2.10 Hypersensitivity to plicamycin (mithramycin) 
2.1.2.11 Requirement for any of the contraindicated medications: nonsteroidal anti-inflammatory 
drugs, aspirin, dextran or other iron containing solutions (due to incompatibility), 
dipyridamole, sulfinpyrazone or valproic acid 
2.1.2.12 Patients who in the opinion of the investigator may not be able to comply with the 
safety monitoring requirements of the study. 
2.1.2.13 Patients receiving concurrently other therapies directed at their cancer. 
2.1.3 Recruitment Strategies 
The following recruitment strategies will be employed to elicit potential candidates for this trial: 
1. Listed on clinical trials.gov;  
2. Listed in PDQ;  
3. In addition, sarcoma patient advocacy and research groups such as the Children’s Oncology 
Group, the Sarcoma Alliance for Research through Collaboration (SARC), The Sarcoma 
Foundation of America, The Liddy Shriver Sarcoma Initiative, the Quad W foundation will 
be notified. 
Prior to distribution of any recruitment materials, such materials will be submitted to the NCI IRB 
for review. 
2.2 
SCREENING EVALUATION 
2.2.1 General 
Pre-treatment blood test should be performed within 7 days and imaging studies within 2 
weeks prior to enrollment on the trial unless otherwise stated.  The evaluations required 
prior to starting treatment are listed in table form in Appendix 3 Study Calendar. 
HISTORY AND PHYSICAL EXAMINATION: including documentation of height and weight 
(average of 3 measurements on the same day), measurable disease, vital signs (blood 
pressure, heart rate, respiratory rate, temperature), performance status, history of bleeding 
disorder, HIV, HBV, HCV, and signs and symptoms within one week of enrollment. 
2.2.2 Laboratory 


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2.2.2.1 ROUTINE LABS: complete blood count, differential, LDH, SGPT (ALT), SGOT (AST), 
alkaline phosphatase, bilirubin (total and direct), BUN, creatinine, amylase, lipase, 
electrolytes, calcium, magnesium, phosphorus, uric acid, albumin and thyroid panel. 
2.2.2.2 Urinalysis. 
2.2.2.3 URINE PREGNANCY TEST: required for females of childbearing potential  
2.2.2.4 Coagulation Studies: PT/PTT/Thrombin Time/Fibrinogen, PFA-100, VWF 
antigen, VWF activity, Factor VIII activity, D-dimer, Fibrin degradation products and FXIII 
activity. In addition, 1 mL of plasma will be obtained, filtered through a 0.22 micron filter, frozen 
and saved for future clinical reference if needed (Error! Reference source not found. and Error! 
Reference source not found.). 
2.2.3 Radiographic Evaluation  
Assessment of disease sites by appropriate radiological evaluation. This could include a CT scan 
of chest, abdomen and pelvis and primary tumor, and bone scan, FDG-PET or MRI as indicated.  
A consistent method of disease evaluation for each patient will be used throughout the study.   
Imaging of the head/brain to rule out intracranial disease including brain metastases is not required 
in all patients, but should be performed if there is clinical concern for intracranial disease/brain 
metastases 
2.2.4 Cardiac Function Studies 
Assessment of cardiac function (ejection fraction/shortening fraction) must be performed within 4 
weeks of enrollment by MUGA or Echocardiogram to rule out cardiomyopathy. In addition, a 12-
lead EKG must be performed. 
2.2.5 Pathologic/Tissue Evaluation 
Histologic confirmation of tumor will be performed on all patients by the NCI Laboratory of 
Clinical Pathology, NIH.  For participants in the Phase II portion:  the presence of the transcript 
with a CLIA approved EWS-FLI paraffin assay will be determined on samples from all sites in the 
Laboratory of Pathology CCR, NCI (See Error! Reference source not found.); unless a pathology 
report documenting presence of the transcript using a CLIA approved assay is obtained from the 
referring institution.   
2.3 
REGISTRATION PROCEDURES 
Patients must be registered by contacting the research nurse in the Pediatric Oncology Branch 
(POB) (When registering a patient, information about all entry criteria (e.g., laboratory results) 
must be available to allow for verification of eligibility. The principal investigator or an Associate 
Investigator must also be contacted to discuss the patient prior to entry on study. Research nurse 
for this trial is: 
Donna M. Bernstein, R.N. 
Pediatric Oncology Branch 
Building 10, Room 13C101 
10 Center Drive 
Bethesda, Maryland 20892  
Phone: (301) 435-7804  
Email: db302w@nih.gov 


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2.3.1 Registration at NCI 
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) 
within 24 hours of signing consent.  A registration Eligibility Checklist from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and faxed to 301-
480-0757.  After confirmation of eligibility at Central Registration Office, CRO staff will call 
pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any 
investigational agents.  Verification of Registration will be forwarded electronically via e-mail to 
the research team. A recorder is available during non-working hours.  
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This study will be conducted as a single institution, open label Phase I/II trial of mithramycin.  The 
Phase I portion will be conducted in children and adolescents (12 months to ≤ 17 years of age) 
with refractory extracranial solid tumors.  The cohort at the recommended dose or MTD will be 
expanded up to 12 patients, and attempts will be made to enroll at least 6 patients that are ≥12 
years of age and 6 patients that are <12 years of age to gain experience with a broad age range of 
patients.   
In the phase II portion of the trial the adult dose (17.5 mcg/kg/dose) will be administered to patients 
with measurable refractory or recurrent Ewing sarcoma and EWS-FLI1 fusion transcript to 
determine the objective response rate (CR and PR) to mithramycin using RECIST criteria.  Initially 
this portion of the trial will be open to eligible participants ≥ 18 years of age.  Enrollment will be 
expanded to include children and adolescents once safety at this dose is determined.  If the pediatric 
phase II dose of mithramycin differs from the adult phase II dose, the protocol will be amended to 
address accrual and response rate to mithramycin in children in a separate stratum. 
The Phase I and Phase II portions of the protocol will enroll patients simultaneously. 
Patients with ESFT enrolled on this study (phase II portion) who meet eligibility criteria for tumor 
biopsies will undergo mandatory tumor biopsies to assess EWS-FLI1 target blockade by 
mithramycin (see Appendix 7: Studies to be performed on tumor tissue). 
Mithramycin will be administered intravenously over 6 hours once daily for 7 days to be repeated 
every 28 days. Patients may continue to receive mithramycin for as long as they tolerate drug 
(Sections 3.1.1 and 3.4) and do not have progressive disease (Section 6.2). 
Patients on study will be closely monitored for the development of toxicity including regular 
physical examinations, blood pressure monitoring, laboratory evaluations, and physical 
examinations with particular attention to the development of any signs of bleeding. Patients will 
be admitted to the hospital for the first treatment cycle during administration of mithramycin in 
order to allow for close monitoring of toxicities. 
Disease will be monitored after every even treatment cycle using appropriate imaging studies using 
RECIST criteria.  
Correlative studies are described in Section 5. 


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3.1.1 Dose Limiting Toxicity 
Dose limiting toxicity (DLT) will be defined as non-hematologic toxicity and hematologic toxicity. 
Toxicities observed during the first treatment cycle will be used to determine the MTD. For the 
Phase II portion of the study, the below toxicities will be considered treatment limiting. 
• Non-hematologic DLT will be defined as any grade ≥ 3 non-hematological toxicity 
possibly, probably, or definitely related to mithramycin with the exception of: 
 Grade 3 nausea that is controlled by symptomatic treatment with anti-emetics 
within 72 hours.  
 Grade 3 vomiting that is controlled by symptomatic treatment with anti-emetics 
within 72 hours.  
 Grade 3 diarrhea that is controlled by symptomatic treatment within 72 hours. 
 Asymptomatic grade 3 elevation of serum transaminases that return to ≤ grade 1 
within 14 days of completing mithramycin administration during a cycle. 
 Asymptomatic grade 3 electrolyte abnormalities that are correctable to grade 2 or 
less within 48 hours.  
• 
Hematologic DLT will be defined as: 
 Grade 4 neutropenia (ANC < 500/mcL) on two consecutive blood counts drawn at 
least 72 hours apart in patients receiving prophylactic growth factor support (See 
Section 3.4) or  
 Grade 4 thrombocytopenia (<25,000/mcL) refractory to platelet transfusion*.  
 Failure to recover platelet count to ≥75,000/mcL by day 42 of starting a treatment 
cycle. 
• 
Any grade 2 bleeding not promptly (within 6 hours of appropriate intervention) corrected 
with blood product support will be considered dose-limiting. 
• 
Bleeding that does not stop within 6 hours of administration of appropriate treatment 
including platelets, will be considered DLT and mithramycin will be discontinued. 
• 
Any grade 2 mithramycin related toxicities of ≥7 days may be considered dose limiting if 
they are intolerable to the patient and cannot be controlled with standard supportive 
measures. 
• 
Failure to recover from toxicity within 42 days of starting a treatment cycle to the level 
required for initiating subsequent cycles (Section 3.4) will be considered dose-limiting 
*Thrombocytopenia will be allowed in this trial provided that that patient is not refractory to 
platelet transfusions and that the bone marrow recovers from the previous cycle.  This toxicity will 
be tolerated because of the theoretical possibility that it reflects mithramycin inhibiting the target.  
It is known that FLI1 plays a prominent role in megakaryocyte development.  Patients with 
haploinsufficiency of FLI1 are known to have congenital thrombocytopenia (Paris-Trousseau 
Syndrome) and megakaryocyte maturation arrest (44).  Furthermore, mithramycin is believed to 
work by blocking binding of FLI1 since that portion of the EWS-FLI1 gene fusion protein is 
responsible for DNA binding.  In addition, patients treated with mithramycin in the 1960s 


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experienced isolated thrombocytopenia.  Therefore, it follows that if mithramycin blocks the action 
of EWS-FLI1, it may also affect FLI1 and therefore cause thrombocytopenia. 
Details regarding the management of thrombocytopenia and bleeding are provided in Section: 4.1 
Details regarding retreatment of patients experiencing neutropenia are provided in Section 3.3. 
3.1.2 Dose Escalation in Phase I Portion 
The initial dose of mithramycin administered in the Phase I portion is 74% of the phase II dose 
which equals 13 mcg/kg/dose, to be repeated every 28 days.  Once safety of the starting dose is 
established, mithramycin will be escalated to the dose of 17.5 mcg/kg/dose according to the 
following table.  
This limited proposed dose escalation is shown below: 
Dose 
Level 
Dose 
(mcg/kg/dose) 
Percent change 
-1 
9.0 
-31% 
1 
13 
0 
2 
17.5 
35% 
All patients will receive dexamethasone as described in Section 4.3. 
Cohorts of 3 to 6 patients will be treated at each dose level.  Toxicity will be graded using version 
4.0 of the NCI Common Toxicity Criteria, and the MTD will be defined based on toxicities 
observed during the first treatment cycle.  When a minimum of 3 patients who are evaluable for 
toxicity have completed 1 cycle of therapy without evidence of DLT, subsequent patients may be 
enrolled at the next higher dose level.  If DLT is observed in 1 patient from the initial cohort of 3 
patients, up to an additional 3 patients will be entered at that dose level.  If none of these additional 
patients experiences a DLT (1/6 with DLT), the dose will be escalated.  If ≥1 of the additional 
patients experience a DLT (≥2/6 with DLT), the MTD has been exceeded. If the MTD has been 
exceeded at the first dose level, then the subsequent cohort of patients will be treated at dose level 
-1.  If ≥2/6 patients experience DLT at the -1 dose (9.0 mcg/kg/dose), no further patients will be 
enrolled until completion of discussions with the sponsor, FDA and NCI IRB and approval of a 
protocol amendment. 
The MTD will be the maximum dose at which fewer than one-third of patients experience DLT 
during cycle 1 of therapy.  
3.1.3 Phase II Portion  
Using a Simon two stage design (see Section 8), mithramycin will be administered intravenously 
at the recommended dose and schedule up to 24 patients evaluable for response.  All patients will 
receive dexamethasone as described in Section 4.3.  Initially adults (≥ 18 years of age) with Ewing 
sarcoma and EWS-FLI1 fusion transcript will be enrolled in order to determine the objective 
response rate (PR and CR) using RECIST. Once adult dose is deemed safe, children (≥ 12 months) 
and adolescents (≤ 17 years) with Ewing sarcoma and EWS-FLI1 fusion transcript will begin 
enrollment to the Phase II portion. Should the pediatric phase II dose of mithramycin differ from 
the adult phase II dose, the protocol will be amended to address accrual and response rate to 
mithramycin in children in a separate stratum. 


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Assessment of NR0B1 in serial tumor biopsies: Should less than 12 patients with evaluable tumor 
biopsies be available at the time response objectives are met, enrollment on this trial will continue 
for patients undergoing tumor biopsies until a minimum of 12 patients with tumor biopsies 
evaluable for analysis of NR0B1 inhibition have been enrolled (Appendix 7: Studies to be 
performed on tumor tissue).  
3.2 
DRUG ADMINISTRATION 
Mithramycin will be administered intravenously during hospitalization for the first cycle. The dose 
of mithramycin will be diluted in appropriate fluids and will be infused over 6 hours daily for 7 
days to be repeated every 28 days  (+/- 2 days) (Appendix 3: Study Calendar).  In the phase II 
portion, mithramycin will be administered at a dose of 17.5 mcg/kg/dose. 
Intravenous hydration should be provided as clinically indicated. 
3.3 
DOSE REDUCTION OR MODIFICATIONS 
Patients who experience a DLT (as defined in Section 3.1.1) possibly, probably or definitely 
related to mithramycin, will have mithramycin held.   
Patients may continue on study at a reduced dose (reduced one dose level as defined in Section 
3.1.2), after having recovered from the experienced DLT within ≤ 42 days of starting the treatment 
cycle.   
If a patient experiences any of the following events, mithramycin will be held and no further 
mithramycin will be administered:  
• grade 3 or 4 anaphylaxis or allergic reaction  
• grade 4 DIC  
• grade 4 erythema multiforme, toxic epidermal necrosis (TEN), or Stevens-Johnson 
syndrome  
• grade 4 diarrhea  
• grade 3 hemorrhage if endoscopic or operative intervention required or any grade 4 
hemorrhage  
• grade 2 or greater bleeding that does not resolve within 6 hours of administration of 
appropriate treatment  
• CNS hemorrhage of any grade   
For the Phase 1 portion only: Patients who initiate treatment at the -1 dose level and experience a 
DLT, will have a ~30% dose reduction for subsequent cycles.  
For both Phase 1 and Phase 2 portions: If another DLT is observed on the reduced dose, the patient 
will discontinue protocol therapy permanently unless the patient is receiving benefit from 
mithramycin as evidenced by disease stabilization or tumor shrinkage, in which case a further dose 
reduction will be permitted.   
Patients who experience Grade 4 neutropenia (<500/mcL) on two consecutive blood counts drawn 
at least 72 hours apart, who recover to meet criteria for additional cycles may receive mithramycin 
at full dose in the next cycle with growth factor support.  If grade 4 neutropenia (<500/mcL) on 
two consecutive blood counts drawn at least 72 hours apart recurs with growth factor support, this 
will be dose-limiting (Section 3.1.1), and dosing may continue at a reduced dose of mithramycin 
as described above with growth factor support.  


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Patients who do not recover from toxicity within 42 days of starting a treatment cycle to the level 
required for initiating subsequent cycles (Section 3.4) will be permanently removed from treatment 
with mithramycin. 
For the Phase 2 portion only: If 2 or more of the initial 6 patients enrolled at the 17.5 mcg/kg dose 
experience dose-limiting hepatotoxicity, enrollment will be put on hold until a discussion with the 
sponsor and IRB and possibly FDA have been held. 
3.4 
CRITERIA FOR ADDITIONAL CYCLES 
Patients will be eligible to continue to receive mithramycin for as long as they do not experience 
clinical or radiographic disease progression and tolerate mithramycin (defined as not meeting off 
treatment criteria).  If a patient experiences DLT on a reduced dose of mithramycin (as defined in 
Section 3.3), no further mithramycin will be administered. 
Patients may receive the next cycle at the same dose level (or reduced level as above) if: 
1) patient has stable or responding disease; 
2) has a platelet count ≥ 75,000/mcL, and an ANC ≥ 1000/mcL, and meets other laboratory 
parameters defined in the eligibility section; and 
3) has not met any criteria for removal from treatment or off study (Section 3.7) 
3.5 
On Study Protocol Evaluation (APPENDIX 3: STUDY CALENDAR) 
3.5.1 Monitoring During Active Treatment:  
3.5.1.1 History and physical exam: at baseline and weekly.  
3.5.1.2 Weight: at baseline and recommended daily days 2 to 7 each cycle  
3.5.1.3 A 12-lead EKG will be performed day 1 and 7 of each cycle, and day 4 of cycle 1. All 
EKGs should be done prior to drug infusion on that day and as close as possible to the 
time the trough PK is drawn.  Document the time the EKG is performed on the PK sheet 
(Appendix 4: Pharmacokinetic Worksheet for Mithramycin (Phase I and Phase II) 
For nursing staff) 
3.5.1.4 Laboratory evaluation:  
CBC with differential count daily (starting on day 2 to day 7) during mithramycin administration; 
then twice weekly during cycles 1-3. During subsequent cycles: CBC with differential count twice 
weekly during administration, then weekly. 
Chemistry: sodium, potassium, chloride, CO2, creatinine, glucose, BUN, albumin, calcium, 
magnesium, daily (starting on from day 2 to day 7) during drug administration; then twice weekly 
during cycles 1-3. During subsequent cycles chemistries are performed weekly.  
Liver Function Tests: alkaline phosphatase, ALT/GPT, AST/GOT, total bilirubin, total protein, 
daily (starting on day 2 to day 7) during drug administration; then twice weekly during cycles 1-
3. During subsequent cycles chemistries are performed weekly. 
Coagulation Studies: PT/PTT/Thrombin Time, Fibrinogen prior to and once on days 3, 4 or 5 and 
days 7, 8, or 9 of every cycle. In addition, prior to cycle 1 and 2, 1 mL of plasma will be obtained, 
filtered through a 0.22 micron filter, frozen and saved for future clinical reference if needed (See 
Appendix 8: Plasma samples for clinical purpose).  
Pregnancy test for women of child-bearing age: prior to each cycle. 


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Urinalysis: prior to each cycle. 
3.5.1.5 Tumor Measurements:   
Disease evaluations will be will be performed after every EVEN numbered treatment cycle, i.e. 
cycle 2, cycle 4, etc, and when removed from treatment, when feasible:  Assessment of disease 
sites will be performed using the most appropriate radiological evaluation.  This could include a 
CT scan of chest, abdomen and pelvis and primary tumor, bone scan, FDG-PET or MRI as 
indicated.  A consistent method of disease evaluation will be used throughout the study. Imaging 
studies used for response evaluation will be sent to the NCI POB for volumetric analysis as 
secondary endpoint (See Appendix 9: Imaging studies for volumetric analysis of tumor 
burden). 
3.5.2 Monitoring after completion of treatment with mithramycin: 
After completion of therapy, patients will be monitored until recovered from toxicity to baseline 
or stabilized. 
3.5.2.1 History and physical exam weekly 
3.5.2.2 Laboratory evaluation: CBC, Acute, Hepatic and Mineral panels, uric acid, LDH, 
PT/PTT weekly 
3.5.3 Off Study Evaluation (at least 30 days after last dose)  
3.5.3.1 History and physical exam  
3.5.3.2 12-lead EKG 
3.5.3.3 Laboratory evaluation: CBC, Acute, Hepatic and Mineral panels, uric acid, LDH, 
PT/PTT 
The patient may have evaluations and lab work obtained through his or her local physician between 
cycles or during follow up. 
3.6 
CONCURRENT THERAPIES 
Other cancer chemotherapy, radiation, immunotherapy, biologic therapy, or investigational agents 
cannot be administered to patient while receiving mithramycin.  
3.7 
 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
3.7.1 Criteria for removal from protocol therapy 
1. A patient may be removed from the protocol for the following non-medical or 
administrative reasons: 
a. Patient refusal of further treatments (reasons must be noted on the patient’s 
CRF). 
b. It is deemed in the best interest of the patient.  In this instance the PI should 
be notified and the reasons of withdrawal should be noted in the CRF. 
c. Serious protocol violation as determined by the PI. 
2. 
Any patient who develops DLT and does not recover from the toxicity by day 42 after 
the start of the cycle (Section 3.1.1 and Section 3.4) 
3. 
Any patient who develops any one of the following: 
a. grade 3 or 4 anaphylaxis or allergic reaction;  
b. grade 4 DIC; 


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c. grade 4 erythema multiforme, toxic epidermal necrosis (TEN), or Stevens-
Johnson syndrome); 
d. grade 4 diarrhea; 
e. grade 3 hemorrhage if endoscopic or operative intervention required or any 
grade 4 hemorrhage;  
f. grade 2 or greater bleeding that does not resolve within 6 hours of 
administration of appropriate treatment; 
g. CNS hemorrhage of any grade. 
4. 
Any patient who does not recover from the toxicity by day 42 to meet criteria to start 
additional cycles (Section 3.4). 
5. 
Any patient who develops DLT after a dose reduction for DLT (Section 3.3) unless the 
patient continues to receive benefit from mithramycin. Patients who received initial 
treatment at 25mcg/kg will be allowed two dose reductions prior to coming off 
treatment. 
6. 
Any patient with clinical or radiographic evidence of progressive disease on treatment, 
as defined in Section 6.2, following any treatment cycle will be removed from the 
treatment.  
7. 
A patient who develops a concurrent serious medical condition that might preclude or 
contraindicate the further administration of mithramycin will be removed from 
treatment.   
8. 
A patient who becomes pregnant will be immediately taken off therapy.  
9. 
Patients who are taken off protocol therapy due to toxicity are to be followed, if 
feasible, until toxicity resolves to baseline or grade 1 or less.  Follow-up data will be 
collected until off study criteria are met unless consent is withdrawn.   
3.7.2 Criteria for removal from Study 
1. 
Death 
2. 
Lost to follow-up 
3. 
Withdrawal of consent for any further data submission 
4. 
Entry onto another therapeutic study 
5. 
Patients who are taken off treatment for toxicity will be followed until resolution of 
toxicity and then taken off study (at least 30 days after the last dose of mithramycin). 
6. 
The PI deems it is in the best interest of the patient 
3.7.3 Off Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off-
study. An off-study form from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm ) main page must be completed and 
faxed to 301-480-0757. 
4 
CONCOMITANT MEDICATIONS AND SUPPORTIVE MEASURES 
4.1 
MANAGEMENT OF BLEEDING, HEMATOLOGIC AND BLOOD PRODUCT SUPPORT 
Blood product support should be provided to maintain platelets ≥ 25,000 cells/mcl and as clinically 
indicated.  
Growth factor support to patients who experience grade 4 neutropenia (ANC <500/µL) will not be 
administered prophylactically, but may be administered as defined in Section 3.3. In addition, 


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growth factor support may be administered to patients who develop grade 4 neutropenia if 
clinically indicated, for example in patients who develop sepsis. 
If patients experience any signs of bleeding (≤ Grade 2) including, epistaxis, hematuria, 
hematemesis or blood in the stool, the patient will immediately undergo a comprehensive clinical 
evaluation, coagulation profile including PT/PTT/fibrinogen, thrombin time, PFA-100, D-dimer, 
and comprehensive metabolic panel including Ca2+ and ionized calcium.  Any abnormality will be 
corrected as clinically indicated with blood product support with guidance by hematology (NIH 
CC hematology consult).  If a patient experiences bleeding in the setting of a normal platelet count, 
platelet function tests will be performed and the patient should be transfused with platelets even in 
the presence of a normal platelet count while these studies are pending.  Mithramycin has also 
been known to block SP1, and SP1 has been shown to play a role in the expression of GPIIb. 
Therefore, mithramycin may cause a deficiency in this protein in mature platelets theoretically 
mimicking the bleeding diathesis, Glanszmanns thrombasthenia (45).  
4.2 
ANTIEMETIC THERAPY 
Ondansetron (or other drugs from this same class of anti-emetics, as per investigator preference) 
prior to and after mithramycin as needed.  Additional antiemetic therapy may be provided by either 
intravenous or oral route as needed.  Patients that experience significant nausea during cycle one 
may be offered aprepitant.  
4.3 
HEPATOTOXICITY PROPHYLAXIS USING DEXAMETHASONE 
Patients will receive 2.5 mg dexamethasone/m2 BSA PO or IV starting 12-16 hours before the 
first dose of mithramycin, then prior to the first dose of mithramycin and then twice daily until24 
hours after the last dose of mithramycin (46, 47).   
4.4 
ANTIDIARRHEALS 
Antidiarrheal agents will be prescribed using standard clinical practice guidelines at the preference 
of the investigator. 
4.5 
ELECTROLYTE REPLACEMENT 
Electrolyte replacement will be provided to maintain serum levels within normal limits. 
4.6 
QTC PROLONGATION 
If grade 3 or 4 QTc prolongation is noted on a 12-lead EKG, then repeat the 12-lead EKG to 
confirm finding and consult cardiology.  Hold next dose until cardiology consult is obtained. 
5 
CORRELATIVE STUDIES / BIOSPECIMEN COLLECTION 
5.1 
PHARMACOKINETIC (PK) SAMPLING 
Detailed plasma pharmacokinetic (PK) sampling of mithramycin will be performed prior to and at 
the completion of the first dose of mithramycin during cycle 1 on the phase I and phase II portion 
of the trial.  In addition, trough and end of infusion samples will be obtained with the day 2, 4 and 
7 doses to assess mithramycin accumulation, and samples will be obtained 24 and 48 hours after 
the day 7 dose of mithramycin on days 8 and 9, respectively.  Deviations in the timing of the PK 
samples will be recorded on the PK worksheets, but not reported as a protocol deviation. In 
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to the NCI IRB.  PK analysis will be performed at the NCI, POB, PETS.  Refer to details in 
Appendix 4: Pharmacokinetic Worksheet for Mithramycin (Phase I and Phase II) For 
nursing staff and Appendix 5: Pharmacokinetic Worksheet for Mithramycin (Phase I and 
Phase II) For Laboratory Personnel and Research Nurses.  
5.2 
STUDIES ON TUMOR TISSUE 
5.2.1 Evaluation of NR0B1 expression on archival tumor tissue  
In all patients with Ewing sarcoma and archival tumor tissue available, evaluation of NR0B1 as 
target of mithramycin will be evaluated using immunohistochemistry in the Laboratory of 
Pathology by Dr. Maria Tsokos and Dr. Mark Raffeld (See Appendix 7: Studies to be performed 
on tumor tissue for details). 
5.2.2 Confirmation of EWS/FLI1 fusion transcript for eligibility for phase II portion: 
Presence of the EWS-FLI1 fusion transcript will be determined during histologic confirmation of 
disease with a CLIA approved EWS-FLI paraffin assay in the Laboratory of Pathology CCR, NCI, 
unless a pathology report documenting presence of the transcript using a CLIA approved assay is 
obtained from the referring institution (See Appendix 7: Studies to be performed on tumor 
tissue for details).  If archival tumor tissue is not available in paraffin blocks, 5 consecutive 
unstained slides and an H& E slide may be used. 
5.2.3 Evaluation of NR0B1 expression from mandatory tumor biopsies  
In patients with Ewing sarcoma and EWS-FLI1 fusion transcript ≥ 18 years old, who have disease 
amenable to percutaneous biopsy, and who meet other eligibility criteria for biopsy, a biopsy will 
be obtained prior to treatment and at steady state on treatment. Patients who meet these criteria 
will have to agree to the biopsies in order to being able to participate in the trial. 
Tissue will be collected and immediately sectioned, a portion will be separated and frozen on dry 
ice for RNA collection for microarray and/or gene signature evaluation of EWS-FLI1 downstream 
target expression (see Section 5.2.3.1).  Another portion will be imbedded in paraffin and 
sectioned.  This tissue will be stained using immunohistochemistry for NR0B1 and if tissue 
remains, proteins in the apoptotic pathway will be evaluated including survivin (BIRC5), XIAP, 
Caspase-8 (CASP8), and BCL-XL (BCL2L1) (See Appendix 7: Studies to be performed on 
tumor tissue for details).  
5.2.4 Other Investigations (See Appendix 7: Studies to be performed on tumor tissue) 
5.2.4.1 RNA for EWS-FLI1 gene signature 
When remaining tissue is available from tumor biopsies pre treatment and at steady state, EWS-
FLI1 gene signature analysis will be performed on the fluidigm platform. 
5.2.4.2 Microarray expression profiling 
When tissue is available, microarray expression profiling using standard Affymetrix HU.133 
platform. 
5.2.4.3 Establishment of Cell Lines 
Finally, if adequate tissue is available and participants agree, efforts will be made to establish cell 
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5.2.5 Future Research Studies 
5.2.5.1 Serum samples for potential biomarker analysis: One serum sample will be obtained 
prior to cycle 1, and then at the same time of restaging studies after every even cycle. 
Details regarding sample handling and time points are provided in Appendix 6: Serum 
Sample For Potential Biomarker (Phase I and Phase II).  
5.3 
SAMPLE STORAGE, TRACKING AND DISPOSITION 
5.3.1 Pediatric Research Samples: Blood Drawing Limits for Research Purposes  
Research blood sample aliquot size will be minimized for patients < 18 years of age and the total 
amount restricted to a maximum of 3 ml/kg per draw and 7 ml/kg per 6-week period (maximum 
450 ml).  If the volume of blood needed for research samples exceeds the sample limits described 
here, pharmacokinetic samples have priority over the serum samples for future biomarker analysis.   
5.3.2 NIH Policy on Research Use of Stored Human Samples, Specimens, or Data 
This study is coordinated by the Pediatric Oncology Branch, NCI.  Sample collection and initial 
processing will be conducted as outlined in Appendices 4 through 8. Samples will only be labeled 
with study identification numbers; no personally identifiable information should be included. 
Storage of pharmacokinetic and pharmacodynamic samples will occur in designated monitored 
freezers (-80°C) in the Pharmacology & Experimental Therapeutics Section (PETS) Laboratory, 
POB, NCI on 1 West.  Imaging studies will be stored at the PETS laboratory on password protected 
computers. All samples obtained on this study will be tracked using LabMatrix.  Pharmacokinetic 
samples will be analyzed at the NCI PETS.  Samples will be identified and tracked using unique 
identifiers linked to each subject's unique patient number (study number).  Codes linking personal 
identifiable information to the unique identifier will be stored in secure, computer servers with 
limited coded access or locked file cabinets in POB with access limited to the PI or study 
coordinator. Archival tumor tissue (blocks or unstained slides will be stained for NR0B1 and other 
markers at the Laboratory of Pathology by Dr. Tsokos.  Unused material will be returned to the 
submitting institution upon their request.  Mandatory tumor samples will be analyzed for NR0B1 
expression at the Laboratory of Pathology.  Gene expression signatures will be analyzed in Dr. 
Helman’s laboratory.  Samples will be stored at -80°C in the laboratory of Dr. Lee Helman.  
Samples will be tracked and considered the responsibility of the principal investigator.  The study 
will remain open and status reported to the NCI IRB until all samples have been analyzed, reported 
or destroyed.  Unintentional loss or destruction of any samples will be reported to the NCI IRB as 
part of annual continuing reviews.  Any use of samples not outlined in Section 5 will require 
prospective NCI IRB review and approval. 
At the termination of this protocol, if additional studies are to be performed on any samples 
retaining patient identifiers, obtained during the conduct of this trial, a Request to Conduct 
Research for Stored Human Samples Specimens, or Data Collected in a Terminated NCI-IRB 
Protocol will be submitted.  Otherwise, specimens will be disposed of in accordance with the 
environmental protection laws, regulations and guidelines of the Federal Government and the State 
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6 
DATA COLLECTION AND EVALUATION  
6.1 
DATA COLLECTION 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The principal 
investigator, associate investigators/research nurses and/or a contracted data manager will assist 
with the data management efforts.  All data obtained during the conduct of the protocol will be 
kept in secure network drives or in approved alternative sites that comply with NIH security 
standards. Primary and final analyzed data will have identifiers so that research data can be 
attributed to an individual human subject participant. 
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breech in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified.  
Patients will complete a patient log for reactions and symptoms related to mithramycin infusion 
(Appendix 10: Patient Log for Reactions/Symptoms), and a concomitant medication log 
(Appendix 11: Patient Log for Concomitant Medication). Physicians or care providers, who 
perform on study evaluations between cycles will receive instructions regarding required on study 
evaluations (Appendix 12:  Monitoring Toxicities Between Mithramycin Cycles with Local 
Physician). 
6.1.1 Source Documents 
Source documents are original documents, data and records which include hospital records, clinic 
and office charts, laboratory data/information, patient diaries or evaluation checklists, pharmacy 
dispensing and other records, recorded data from automated instruments, microfiches, 
photographic negatives, microfilm or magnetic media, x-rays.  The Principal Investigator will 
permit trial-related monitoring, audits, IRB review, and regulatory inspections, providing direct 
access to source data documents.  
6.2 
RESPONSE CRITERIA 
Objective response and progression will be evaluated in this study using the new international 
criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) Committee 
[version 1.1 (48)].   
Note:  Lesions are either measurable or non-measurable using the criteria provided below. The 
term “evaluable” in reference to measurability will not be used because it does not provide 
additional meaning or accuracy.    
6.2.1 Response Criteria for Radiographic Studies 
6.2.1.1 Measuring of Soft Tissue Disease 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
[version 1.1 (48)].  Changes in the largest diameter (unidimensional measurement) of the tumor 
lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST 
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a. 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 the diameters of target lesions, taking as 
reference the baseline sum diameters 
 
 
Progressive Disease (PD)  
At least a 20% increase in the sum of the diameters of target lesions, taking as reference 
the smallest sum on study (this includes the baseline sum if that is the smallest on 
study).  In addition to the relative increase of 20%, the sum must also demonstrate an 
absolute increase of at least 5 mm.  (Note:  the appearance of one or more new lesions 
is also considered progressions) 
 
 
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 
b. Evaluation of Non-Target Lesions  
 
 
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) 
Note:  If tumor markers are initially above the upper normal limit, they must normalize 
for a patient to be considered in complete clinical response. 
 
 
Non-CR/Non-PD (Stable Disease, SD) 
Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker 
level above the normal limits. 
 
 
Progressive Disease (PD)  
Appearance of one or more new lesions and/or unequivocal progression of existing 
non-target lesions. Unequivocal progression should not normally trump target lesion 
status.  It must be representative of overall disease status change, not a single lesion 
increase.     
 Although a clear progression of “non-target” lesions only is exceptional, the opinion 
of the treating physician should prevail in such circumstances, and the progression 
status should be confirmed at a later time by the review panel (or Principal 
Investigator). 
c. Evaluation of Best Overall Response 
 The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best 


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response assignment will depend on the achievement of both measurement and 
confirmation criteria. 
Target Lesions 
Non-Target 
Lesions 
New Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR 
Non-PD 
No 
PR 
 
>4 wks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR 
Non-PD 
Not evaluated 
No 
PR 
SD 
Non-CR 
Non-PD 
Not evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or No 
PD 
 
No prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
      *See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
          **Only for non-randomized trials with response as primary endpoint. 
         ***In exceptional circumstances, unequivocal progression in non-target lesions may be 
accepted as disease progression 
 
d. 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 
the objective progression even after discontinuation of treatment. 
e. In some circumstances, it may be difficult to distinguish residual disease from normal 
tissue.  When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesions be investigated (fine needle aspirate/biopsy) 
before confirming the complete response status. 
6.2.2 Confirmatory Measurement/Duration of Response 
a. Confirmation 
To be assigned a status of PR or CR, changes in tumor measurements must be 
confirmed by repeat assessments that should be performed at least 4 weeks after the 
criteria for response are first met.  Objective response measures will be confirmed at 
the Coordinating Center.  The participating site should forward staging studies of 
participants who have achieved PR or CR status to the Coordinating Center for 
confirmation. 
b. Duration of Overall Response 
The duration of overall response is measured from the time measurement criteria are 
met for CR or PR (whichever is first recorded) until the first date that recurrent or 


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progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first 
met for CR until the first date that progressive disease is objectively documented. 
6.2.3 Measurable Disease 
Measurable lesions are defined as those that can be accurately measured in at least one dimension 
(longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm with CT scan, or >10 mm 
with calipers by clinical exam.  All tumor measurements must be recorded in millimeters (or 
decimal fractions of centimeters). 
Note:  Tumor lesions that are situated in a previously irradiated area might or might not be 
considered measurable.  If the investigator thinks it appropriate to include them, the conditions 
under which such lesions should be considered must be defined in the protocol. 
6.2.4 Malignant Lymph Nodes  
To be considered pathologically enlarged and measurable, a lymph node must be >20 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. 
6.2.5 Non-Measurable Disease 
All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or 
pathological lymph nodes with ≥ 10 to <15 mm short axis), are considered non-measurable disease.  
Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or 
MRI), are considered as non-measurable. 
Note:  Cystic 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. 
6.2.6 Target Lesions 
All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative 
of all involved organs, should be identified as target lesions and recorded and measured at 
baseline.  Target lesions should be selected on the basis of their size (lesions with the longest 
diameter), be representative of all 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.  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 
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. 


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Progressive disease by RECIST criteria [1] noted after the first re-staging scan may represent 
disease that was not detected on the pre-study scan, and a confirmatory scan will be required at the 
next scheduled re-staging evaluation unless clinically not indicated.  If confirmed, progression 
should be dated by the initial time when the lesions are first detected. If progressive disease by 
RECIST criteria is seen after cycle 2, but not confirmed on subsequent restaging scan, the scans 
from after cycle 2 would serve as the baseline scan to evaluate for disease progression (49). 
6.2.7 Non-Target Lesions 
All other lesions (or sites of disease) including any measurable lesions over and above the 5 target 
lesions should be identified as non-target lesions and should also be recorded at baseline. 
Measurements of these lesions are not required, but the presence, absence, or in rare cases 
unequivocal progression of each should be noted throughout follow-up.  
6.2.8 Metastatic Bone Lesions 
Disease progression is considered if a minimum of two new lesions is observed on bone scan. New 
lesions seen by the end of cycle 2 or before cycle 3 (with the first re-staging bone scan) may 
represent disease that was not detected on the pre-study scan, and a confirmatory scan will be 
required at the next scheduled re-staging bone scan unless clinically not indicated.  If confirmed, 
progression should be dated by the initial time when the lesions are first detected. If new lesions 
are seen after cycle 2, but no additional lesions are seen on confirmatory scans, the scans from 
post-cycle 2 would serve as the baseline scan to evaluate for disease progression (49). 
Clinical Lesions  
Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules 
and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers (e.g., skin nodules).  
In the case of skin lesions, documentation by color photography, including a ruler to estimate the 
size of the lesion, is recommended.  
6.2.9  Guidelines for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment. 
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 is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
6.2.10  Methods of Measurement 
Chest X-ray - Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable.  
CT and MRI - CT and MRI are the best currently available and reproducible methods to measure 
target lesions selected for response assessment. For this study helical Multi-detector CT will be 
performed with cuts of 5 mm in slice thickness for chest, abdomen and pelvis lesions and 2-3 mm 
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6.2.11 Additional response evaluation using volumetric analysis 
In addition, the utility of volumetric tumor measurement in patients with measurable disease will 
be prospectively evaluated and compared to 1D and 2D measurements.  All sites will submit 
baseline and restaging studies to NCI for volumetric analysis.  (See Error! Reference source not 
found. for details). 
6.3 
TOXICITY CRITERIA  
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting.  All appropriate treatment areas have access to a copy of the CTCAE version 4.0.  A 
copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov). 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
An adverse event is defined as any reaction, side effect, or untoward event that occurs during the 
course of the clinical trial, whether or not the event is considered related to the treatment or 
clinically significant. For this study, AEs will include events reported by the patient, as well as 
clinically significant abnormal findings on physical examination or laboratory evaluation. A new 
illness, symptom, sign or clinically significant laboratory abnormality or worsening of a pre-
existing condition or abnormality is considered an AE.  All AEs must be recorded on the AE case 
report form.   
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days 
after the last administration of investigational agent/intervention and have an attribution of at 
least possibly related to the agent/intervention should be recorded and reported as per sections 
7.2, 7.3, and 7.4.  
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms  
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization 
• 
Is judged by the Investigator to be of significant clinical impact 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
7.1.2 Suspected adverse reaction 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 


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possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
7.1.3 Unexpected adverse reaction 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the 
investigator brochure or is not listed at the specificity or severity that has been observed; or, if an 
investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application. "Unexpected”, 
also refers to adverse events or suspected adverse reactions that are mentioned in the investigator 
brochure as occurring with a class of drugs or as anticipated from the pharmacological properties 
of the drug, but are not specifically mentioned as occurring with the particular drug under 
investigation. 
7.1.4 Serious 
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious 
Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 
7.1.5 Serious Adverse Event (SAE) 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following: 
• 
Death 
• 
A life-threatening adverse drug experience 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions 
• 
A congenital anomaly/birth defect 
• 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition 
7.1.6 Disability 
 A substantial disruption of a person’s ability to conduct normal life functions. 
7.1.7 Life-threatening adverse drug experience 
Any adverse event or suspected adverse reaction that places the patient or subject, in the view of 
the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that had it occurred in a more severe form, might have caused death. 
7.1.8 Protocol Deviation (NIH Definition) 
Any change, divergence, or departure from the IRB approved research protocol. 


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7.1.9 Non-Compliance (NIH Definition) 
The failure to comply with applicable NIH Human Research Protections Program (HRPP) 
policies, IRB requirements, or regulatory requirements for the protection of human research 
subjects. 
7.1.10 Unanticipated Problem 
Any incident, experience, or outcome that: 
•  Is unexpected in terms of nature, severity, or frequency in relation to  
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and 
(b) the characteristics of the subject population being studied; AND 
• Is related or possibly related to participation in the research; AND 
• Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
7.2 
NCI-IRB AND NCI CLINICAL DIRECTOR REPORTING 
7.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report in the NIH Problem Form to the NCI-IRB and NCI Clinical Director: 
• All deaths, except deaths due to progressive disease  
• All Protocol Deviations 
• All Unanticipated Problems 
• All non-compliance 
Reports must be received within 7 working days of PI awareness via iRIS. 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review 
The protocol PI will report to the NCI-IRB: 
1. A summary of all protocol deviations in a tabular format to include the date the deviation 
occurred, a brief description of the deviation and any corrective action. 
2. A summary of any instances of non-compliance 
3. A tabular summary of the following adverse events: 
• 
All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research;  
• 
All Grade 3 and 4 events that are possibly, probably or definitely related to the research;  
• 
All Grade 5 events regardless of attribution;  
• 
All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.  


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7.2.3 NCI-IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NCI IRB. 
7.2.4 Request for waiver from IRB reporting: Anticipated PD and Expedited Non-UP AEs 
Protocol evaluations and initiation of mithramycin treatment for subsequent cycles, which are 
performed within a window of +/- 7 days of the protocol required schedule will not be 
considered a deviation, and not reported to the IRB. 
7.3 
IND SPONSOR REPORTING CRITERIA  
7.3.1 Expedited Adverse Event Reporting Criteria to the IND Sponsor 
An investigator must immediately report to the sponsor, using the mandatory MedWatch form 
3500a, any serious adverse event, whether or not considered drug related, including those listed 
in the protocol or investigator brochure and must include an assessment of whether there is a 
reasonable possibility that the drug caused the event.  
• All Grade 5 (fatal) events (except death due to progressive disease) must be reported via 
email within 24 hours.  A complete report must be submitted within one business day.  
• All other serious adverse events including deaths due to progressive disease must be 
reported within one business day 
Study endpoints that are serious adverse events (e.g. all-cause mortality) must be reported in 
accordance with the protocol unless there is evidence suggesting a causal relationship between 
the drug and the event (e.g. death from anaphylaxis).  In that case, the investigator must 
immediately report the death to the sponsor. 
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov 
7.3.2 Annual Reporting of AEs to the Sponsor 
 
7.4 
FDA REPORTING CRITERIA 
7.4.1 IND Safety Reports to the FDA (Refer to 21 CFR 312.32) 
The Sponsor will notify the FDA of any unexpected fatal or life-threatening suspected adverse 
reactions as soon as possible but no later than 7 calendar days of initial receipt of the information 
using the MedWatch Form 3500a.  
The Sponsor is also responsible for reporting:  
• any suspected adverse reaction that is both serious and unexpected; 
• any findings from clinical, epidemiological, or pooled analysis of multiple studies or any 
findings from animal or in vitro testing that suggest a significant risk in humans exposed 
to the drug; 
• clinically important increase in the rate of a serious suspected adverse reaction over that 
listed in the protocol or investigator brochure;  
to the FDA and to all investigators no later than 15 calendar days after determining that the 
information qualifies for reporting using the MedWatch Form 3500a. If FDA requests any 


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additional data or information, the sponsor must submit it to the FDA as soon as possible, but no 
later than 15 calendars days after receiving the request. 
7.4.2 FDA Annual Reports (Refer to 21 CFR 312.33) 
The study Sponsor will submit a brief report annually of the progress of the trial within 60 days 
of the anniversary date that the IND went into effect as indicated in 21CFR 312.33, and any 
associated FDA correspondences regarding the IND annual report.   
7.5 
DATA AND SAFETY MONITORING PLAN 
7.5.1 Principal Investigator/Research Team  
The clinical research team will meet on a regular basis, biweekly when patients are being actively 
treated on the trial to discuss each patient in detail.  The PI will meet regularly with participating 
site investigators during study enrollment and treatment.  Decisions about dose level enrollment 
and dose escalation during the phase I portion will be made based on the toxicity data from prior 
patients.  
All data will be collected in a timely manner, entered into C3D and reviewed by the principal 
investigator or a lead associate investigator in a timely manner.  Adverse events will be reported 
as required above. Any safety concerns, new information that might affect either the ethical and 
or scientific conduct of the trial, or protocol deviations will be immediately reported to the IRB 
using iRIS and to the Sponsor.  
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy.  The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff.  
7.5.2 Sponsor Monitoring Plan 
This trial will be monitored by personnel employed by Harris Technical Services on contract to 
the NCI, NIH.  Monitors are qualified by training and experience to monitor the progress of clinical 
trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct. 
At least 25% of enrolled patients will be randomly selected and monitored at least biannually, 
based on accrual rate.  The patients selected will have 100% source document verification done. 
Additional monitoring activities will include: adherence to protocol specified study eligibility, 
treatment plans, data collection for safety and efficacy, reporting and time frames of adverse events 
to the NCI IRB and FDA, and informed consent requirements.  Written reports will be generated 
in response to the monitoring activities and submitted to the Principal investigator and Clinical 
Director or Deputy Clinical Director, CCR, NCI. 
8 
STATISTICAL CONSIDERATIONS   
8.1 
PHASE I PORTION 
The phase I portion of this study will enroll patients in a standard 3 + 3 design as described in 
Section Error! Reference source not found..  The cohort at the recommended dose or MTD will be 
expanded to 12 patients, and attempts will be made to enroll at least 6 patients that are ≥12 years 
of age and 6 patients that are <12 years of age to gain experience with a broad age range of patients. 


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The maximum number of evaluable patients for this phase is 18 (6 patients at 2 dose levels and 6 
to expand MTD to 12).   
8.2 
PHASE II PORTION 
The primary endpoint will be objective response by RECIST criteria v 1.1. The best response of 
disease to mithramycin will be examined. The following outlines the Simon optimal two stage 
design that will be used: 
 
Cumulative 
Number of 
Responses 
Decision 
Stage 1: Enter 9 patients 
0 
Terminate the trial: agent 
ineffective 
 
1 or more 
Inconclusive result, continue trial  
 
 
(proceed to Stage 2) 
Stage 2: Enter 15 
additional patients 
2 or fewer      
Terminate the trial: agent 
ineffective 
 
3 or more 
Terminate the trial: agent effective 
We will consider mithramycin not of sufficient interest for further evaluation in a disease category 
if the true response rate is 5% (p0=0.05) and of sufficient activity if the true response rate is 25% 
(p1=0.25).  As summarized in the above table, with alpha=0.10 (10% probability of accepting an 
ineffective treatment) and beta=0.10 (10% probability of rejecting an effective treatment), the 
study will initially enroll 9 patients and if 0 of 9 have a response, then no further patients will be 
enrolled.  If 1 or more respond, accrual will continue until a total of 24 evaluable patients have 
been enrolled.  After 24 patients have been enrolled and evaluated, 1-2 with a response will be 
considered insufficient while if 3 or more respond in 24 patients, this will be considered sufficient 
activity for further investigation.  Under the null hypothesis, the probability of early termination 
is 63%. 
The two patients that were enrolled in the 25 mcg/kg dose of the Phase II portion will be evaluated 
for response but not be counted as evaluable for the design above, as they received mithramycin 
at a higher single dose (25 mcg/kg) than the new phase II dose (17.5 mcg/dose) determined with 
amendment B. The accrual ceiling will be increased by two patients to allow for these non-
evaluable patients. 
If the pediatric phase II dose of mithramycin determined on this trial differs from the adult phase 
II dose, the protocol will be amended to address accrual and response rate to mithramycin in 
children in a separate stratum. 
8.2.1 Methods of Analysis 
Patients who are considered evaluable for response will be included in an analysis of time to 
progression.  Time to progression will be estimated using the product-limit method of Kaplan and 
Meier. The probability of progression free survival at 6 months will be summarized.  Response 
rates will be calculated as the percent of patients whose best response is a CR or PR, and the 
fraction of responses obtained will have a 95% confidence interval, which takes into consideration 
the two-stage nature of the design (50).  Toxicity information recorded will include the type, 


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severity, time of onset, time of resolution, and the probable association with the study regimen.  
Tables will be constructed to summarize the observed incidence by severity and type of toxicity. 
Time to progression will be taken as the number of days from enrollment until: (1) disease 
progression; (2) death because of treatment complications; (3) resection of measurable tumor; or 
(4) last patient follow-up whichever is first.  Patients will be considered to have experienced a 
progression event if (1) or (2) occurs.  Otherwise, the patient will be considered censored for time 
to progression. 
8.3 
EVALUATION OF RESPONSE  
Any patient who is enrolled and receives at least one dose of mithramycin will be considered 
evaluable for response provided: (1) the patient demonstrates progressive disease or death while on 
protocol therapy; (2) the patient is observed on protocol therapy for at least one cycle and the tumor 
is not removed surgically prior to the time complete response or partial response is confirmed; or (3) 
the patient demonstrates a complete or partial response as confirmed according to protocol criteria.  
Patients who electively terminate therapy before receiving all 7 doses of mithramycin during the 
first treatment cycle and do not expire within 28 days from start of treatment will be replaced.  
Patients who demonstrate a complete or partial response will be considered to have experienced a 
response. The evaluation period for determination of the best response will be 6 treatment cycles. 
All other patients will be considered non-responders.   
8.4 
INHIBITION OF NR0B1 IN TUMOR TISSUE AND CHANGES IN GENE EXPRESSION 
SIGNATURE 
It will be important to determine if mithramycin inhibits NR0B1 in at least a modest fraction of 
patients in order to confirm that the agent is correctly meeting its intended target.  This will require 
that biopsies be obtained on subjects 18 years or older with accessible biopsy sites, at both pre-
treatment as well as on treatment.  Given that no more that 42 subjects are expected to enroll onto 
this study as a maximum, the accrual target for this endpoint will be based on no more than 50% 
of the maximum enrollment projected.  This portion of the study will be conducted as a pilot using 
up to 20 evaluable subjects.  With 20 subjects, there would be 90% power to detect a difference 
between an unacceptably low fraction with inhibition of 5% and a more modestly acceptable 
fraction of 25% using a one-sided 0.10 alpha level exact test for a binomial proportion.  If only 12 
patients are able to provide biopsy results at both times, there would be 90% power to detect a 
difference between 5% and 45% with inhibition.  Thus, while up to 20 patients with paired biopsies 
would be desirable, as few as 12 patients with the complete evaluation would be adequate to 
address this to some degree.  
Should less than 12 patients with evaluable tumor biopsies be available at the time the toxicity and 
response objectives are met, enrollment on this trial will continue for patients undergoing tumor 
biopsies until a minimum of 12 patients with tumor biopsies evaluable for analysis of NR0B1 
inhibition have been enrolled.  In order to be evaluable for analysis of NR0B1 inhibition, NR0B1 
expression has to be present on the tumor biopsy obtained at baseline.  
8.5 
SECONDARY ENDPOINTS  
Pharmacokinetic analysis will be conducted using non-compartmental methods and estimated 
pharmacokinetic parameters will be presented for each dose level using summary statistics.  When 


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pharmacodynamic studies are analyzed, descriptive statistics for each dose level will be calculated 
for each surrogate endpoint.  
The associations and differences between tumor burden and change in tumor burden as measured 
by RECIST, WHO, and volumetric analysis will be summarized with scatter plots and correlation 
coefficients.  The results of these descriptive analyses will be used to design further studies. 
8.6 
NUMBER AND RATE OF ACCRUAL  
If a maximum of 18 patients are required to complete the phase I portion, and 9 to 24 patients are 
required to complete the phase II portion, the maximum number of evaluable patients required to 
complete both portions of this trial is 42.   
With an estimated monthly accrual of 1-2 patients, the projected duration of this study is 2 to 3.5 
years. 
9 
HUMAN SUBJECTS PROTECTIONS  
9.1 
RATIONALE FOR SUBJECT SELECTION 
The Ewing sarcoma family of tumors (ESFT) is the second most common malignant bone tumor 
of childhood.  In the past 25 years since the advent of 5 drug ESFT therapy, there has been very 
little improvement in overall patient survival particularly for patients with high risk metastatic or 
relapsed disease.  Laboratory research has demonstrated potential efficacy of mithramycin in 
Ewing Sarcoma Family of Tumors with EWS-FLI1 fusion transcript. 
No groups, in regards to gender, and racial and ethnic groups, are being excluded from 
participation in the trial.  Females who are pregnant or breastfeeding will not be eligible for the 
trial due to risks of fetal and teratogenic adverse events as seen animal studies. 
9.2 
PARTICIPATION OF CHILDREN 
This study is designed to define the toxicities and maximum tolerated dose of mithramycin in 
children and adolescents with solid tumors.  It will also evaluate the activity of mithramycin in the 
treatment of children and adolescents, as well as adults with Ewing Sarcoma Family of Tumors 
with EWS-FLI1 fusion transcript.  Therefore children and adolescents will be entered onto this 
research study.  Adults (≥ 18 yrs) who are cognitively impaired prior to study entry and who have 
not previously assigned DPA to a family member or friend will not be eligible for the trial, because 
they cannot give informed consent.  Pediatric and adult oncologists who have extensive experience 
in performing investigational drug trials in children will conduct the trial.  Patients will be cared 
for in the POB outpatient pediatric oncology clinic or outpatient setting and if patients require 
hospital admission, they will be cared for on the pediatric unit of the CRC by the POB staff. 
Patients > 35 years old will be cared for by adult oncologists. 
9.3 
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
Patients under the age of 18 years will be entered on this trial.  The primary risk to patients 
participating in this research study is from toxicity of mithramycin, an investigational agent in this 
patient population.  The protocol provides for detailed and careful monitoring of all patients to 
assess for toxicity and the dose escalation scheme is very conservative.  Toxicity data from the 
current dose level will be collected and reviewed to ensure that there were no severe (dose-
limiting) toxicities prior to escalating to the next higher dose level.  Although the primary objective 


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of the phase I portion of this trial is to define the qualitative toxicities and maximum tolerated dose 
of the drug, all patients entered on the trial will have refractory or recurrent disease, which this 
agent may benefit.  Patients enrolled on the phase II portion of the study will be diagnosed with 
Ewing Sarcoma Family of Tumors with EWS-FLI1 fusion transcript, for which laboratory data 
suggested this agent may be of benefit.  Mithramycin offers a potential for direct benefit although 
the likelihood of this may be small.  The potential benefits from this therapy are disease 
stabilization or shrinkage and a reduction in symptoms caused by the tumor.  Therefore, this 
protocol involves greater than minimal risk, but presents the potential for direct benefit to 
individual subjects. 
Mandatory serial tumor biopsies will be obtained in participants with Ewing sarcoma with EWS-
FLI1 fusion transcript who are 18 years of age or older, who have disease amenable to biopsy as 
outlined in Section Error! Reference source not found..  Risks include risk of bleeding, bruising, 
pain or rarely, infection.  
9.4 
CONSENT AND ASSENT PROCESS AND DOCUMENTATION 
The investigational nature and research objectives of this trial, the procedures and treatments 
involved and their attendant risks and discomforts and benefits, and potential alternative therapies 
will be carefully explained to the patient or the patient’s parents or guardian if he/she is a child, 
and a signed informed consent document will be obtained prior to entry onto the study. The 
investigators are requesting a waiver from the IRB to allow only one parent to sign the informed 
consent to enter a child on the protocol.  Because many patients must travel to the NIH from long 
distances at substantial expense, requiring both parents to be present for the consent process could 
be a financial hardship for many families.  The PI or an associate investigator of the trial will 
obtain consent.  Where deemed appropriate by the clinician and the child’s parents or guardian, 
the child will also be included in all discussions about the trial and verbal assent will be obtained.  
The parent or guardian will sign the designated line on the informed consent attesting to the fact 
that the child has given assent.  
10 PHARMACEUTICAL INFORMATION   
10.1 MITHRAMYCIN (PLICAMYCIN) 
Mithramycin is an antineoplastic antibiotic (oligosaccharide) produced by the growth of 
Streptomyces argillaceus, S. plicatus and S. tanashiensis.  Previously used in the treatment of 
inoperable metastatic neoplasms of the testes, and for the symptomatic treatment of hypercalcemia 
and hyercalciuria. 
10.1.1 Source  
Mithramycin was supplied by Fermentek Ltd (Jerusalem, Israel) to the NCI under an IND held by 
the CCR.  The NIH Clinical Center Pharmacy will be testing and vialing the investigational agent. 
Drug will be shipped to participating centers upon receipt of IRB approval.  
10.1.2 Toxicity  
The following effects have been observed in previous human administration (possible signs and 
symptoms in parentheses where appropriate) 
More frequent: 


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• Hypocalcemia (muscle and abdominal cramps) 
• Anorexia (loss of appetite) 
• Diarrhea 
• Stomatitis  
• Nausea or vomiting —may occur 1 to 2 hours after initiation of therapy and continue for 
12 to 24 hours 
Note: Incidence and severity of gastrointestinal side effects may increase with too rapid a 
rate of administration. 
Less frequent 
• Drowsiness  
• Fever  
• Headache 
• Mental depression  
• Pain, redness, soreness, or swelling at injection site  
• Unusual tiredness or weakness http://www.drugs.com/mmx/mithracin.html - 
citec00152801 
• Gastrointestinal bleeding (bloody or black, tarry stools; vomiting of blood) 
• hepatotoxicity (yellow eyes or skin) ALT and AST (severe) and GGT elevation 
• LDH elevation 
• Epistaxis, hematemesis 
• Leukopenia (sore throat and fever)—incidence about 6% 
• Petechial bleeding  
• Thrombocytopenia  
• Toxic epidermal necrolysis (flushing or redness or swelling of face; skin rash)—possible 
early symptoms of overdose 
Note: Hemorrhagic diathesis—Incidence more frequent with doses of more than 30 mcg 
(0.03 mg) per kg of body weight a day and/or for more than 10 doses.  
10.1.3 Formulation and preparation  
Mithramycin is supplied as 2 mg/vial, freeze dried powder for injection.  To prepare the initial 
dilution of 500 mcg of mithramycin per mL, add 3.9 mL of sterile water for injection to the 2 mg 
vial and shake to dissolve.  After the appropriate dose has been withdrawn from the vial, discard 
the unused portion.  
For intravenous infusion, doses should be diluted as follows: 


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Mithramycin dose (mcg) 
Infusion Solution: 0.9% Sodium Chloride or 
5% Dextrose Injection (mL) 
1000 or above 
1000  
500-999  
500 
250-499 
250 
100-249 
100 
All dilutions will be infused over 6 hours. 
10.1.4 Stability and Storage  
Prior to reconstitution, store between 2 and 8 °C (36 and 46 °F).  Store in a light-resistant container. 
Reconstituted solution (500 mcg per mL) should be freshly prepared for each dose and used 
immediately.  Studies in Clinical Center Pharmacy Department indicate that the drug is stable for 
24 hours at room temperature when further diluted in 0.9% Sodium Chloride or 5% Dextrose 
Injection to concentrations between 1 and 25 mcg/ml when protected from light.  Because this is 
a pharmacokinetic study and the drug concentrate does not contain a preservative, infusion 
solutions should be prepared daily within four hours of the scheduled infusion.  Discard any unused 
portion of either solution. 
10.1.5 Administration procedures 
Infuse intravenously over 6 hours using chemotherapy precautions.  Infusion bags have to be 
protected from light from the time of preparation through completion of the infusion. Observe 
frequently for signs of extravasation.  Should extravasation occur, the infusion should be 
terminated at that site and reinstituted at another site.  Moderate heat should be applied to the site 
of extravasation to disperse the drug and minimize local tissue irritation and discomfort.  No in-
line filter should be used for mithramycin administration. 
10.1.6 Contraindications  
The following medications are contraindicated:  
• Anticoagulants, coumarin- or indandione-derivatives  
• Heparin other than heparin flushes 
• Thrombolytic agents  
• Anti-inflammatory drugs, nonsteroidal (NSAIDs) or aspirin or salicylate-containing products, 
which may increase risk of hemorrhage 
• Dextran  
• Dipyridamole  
• Sulfinpyrazone  
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10.2 DEXAMETHASONE 
10.2.1 Source: 
Dexamethasone will be provided from commercial sources by the NIH Clinical Center Pharmacy 
Department.  
10.2.2 Toxicity: 
Common   
Cardiovascular: Hypertension  
Dermatologic: Atrophic condition of skin, Finding of skin healing, Impaired  
Endocrine metabolic: Cushing's syndrome, Decreased body growth  
Gastrointestinal: Disorders of gastrointestinal tract  
Immunologic: At risk for infection  
Musculoskeletal: Osteoporosis  
Ophthalmic: Cataract (5%), Raised intraocular pressure (25%)  
Psychiatric: Depression, Euphoria  
Respiratory: Pulmonary tuberculosis 
Serious   
Endocrine metabolic: Hyperglycemia, Primary adrenocortical insufficiency  
Ophthalmic: Conjunctival hemorrhage (22%), Glaucoma, Vitreous detachment (2%) 
Musculoskeletal and connective tissue disorders: Avascular Necorsis 
10.2.3 Formulation and preparation: 
Oral Tablet (Scored): 4 mg 
Injection, solution, as sodium phosphate:  4 mg/mL (1 mL, 5 mL, 30 mL);  
10.2.4 Administration procedures: 
Oral: Administer with meals to decrease GI upset.  
I.V.: Administer intravenously over 10 minutes.  
10.2.5 Incompatibilities 
Contraindicated:  Praziquantel (theoretical), Rotavirus Vaccine, Live (established) 
Major:  Aldesleukin (theoretical), Bupropion (theoretical), Darunavir (theoretical), Dasatinib 
(theoretical), Etravirine (theoretical), Fosamprenavir (theoretical), Imatinib (theoretical), 
Ixabepilone (theoretical), Lapatinib (theoretical), Nilotinib (theoretical), Quetiapine (probable), 
Romidepsin (theoretical), Sunitinib (theoretical), Temsirolimus (theoretical), Thalidomide 
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11 APPENDICES 
11.1 APPENDIX 1: MITHRAMYCIN CLINICAL EXPERIENCE REPORTED IN THE LITERATURE 
Author 
Year 
Patient Age 
Disease 
Response rate 
Dose 
Schedule 
Toxicity  
In cycle 1 
Toxicity 
subsequent 
cycles 
MTD? 
PK 
F Fraisse (51) 
1980 
26 
Hypercalce
mia in 
pregnancy 
- 
25 mcg/kg  
daily x4 
Day 4 fever 40° C, shock, SGOT 
1400 mUI/L, SGPT 1600 mUI/L, plt 
52,000, hemothorax, diffuse 
hemorrhage, hepatic necrosis, death 
- 
- 
- 
Donaldson(52) 
1985 
NONE 
 
 
 
 
 
 
 
 
Kofman (33) 
“Tx of 
Disseminated.” 
1963 
 
10-74 
(10 yo 
ESFT) 
 4 under 22 
Various 
1 ESFT 
84 pts 
(68 evaluated) 
6 regressions 
2 subjective 
improvements 
25 
ug/kg/day 
as 24 hour 
infusion 
Daily as 
tolerated 
(up to 16) 
-a few got 
24 
Hemorrhage- 3  deaths 
“usually related to 
thrombocytopenia” 
-one patient with bleeding time of 90 
minutes 
-no deaths at 25 ug/kg with 
continuous infusions 
-Nausea, Vomiting, anorexia, restlessne
irritability 
“Hepatic and renal damage not major
factors except in patients who receive
very large doses or who had poor hepa
 
or renal function at start” 
“ a surprising 
factor was 
severe 
cumulative 
toxicity 
-again 
hemorrhage on 
second course 
with PLT >50 
50 ug/kg 
tried 
 
Dutcher (42) 
1997 
35-74 
(median 51) 
CML 
(mithra + 
interferon) 
13 pts 
2SD 
3PR 
1CR 
25 ug/kg 
over 2-4 
hrs  
M, W,F for 
2 weeks 
then one 
dose 
Qmonth 
1 patient removed for 
“hepatotoxicity” 
not defined 
Not defined 
Not 
defined 
 
Thurlimann, B 
(43) 
1992 
27-74 
(median 62) 
Hypercalce
mia 
N/A 
25 ug/kg 
Day 1 
-Vomiting  
Moderate 18% 
Severe 5% 
-Phlebitis 11% 
-Fever 9% 
-Mean 2x AST/ALT 
-DEATH arterial bleed from 
duodenal ulcer 1 Patient 
*thrombocytopenia 5% 
 
 
 
Lebbin, D (32) 
1974 
47-74 yrs 
10 patients 
with Paget’s 
10/10 with 
improved pain 
and increased 
activity 
25 ug/kg 
Daily x 10 
for cycle 1 
Then 
A = Daily 
x5 every 
-N/V – responded to anti-emetics NO 
dose reductions 
-uremia- 7/10  
-thrombocytopenia 1/10 (<150k) 
-no evidence of 
cumulative 
hepatotox. 
 
 


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(all regimens 
effective) 
 
                
week for a 
month 
Vs 
B = Q 
week for 14 
months 
Vs 
B1 = twice 
weekly for 
5 months 
NO hemorrhage-withheld drug if 
PLT<150k 
-Isocitrate Dehydrogenase elevation 
1/10 
 
Johnson, P.R.E 
(53) 
1991 
28-57 
CML in 
blast crisis 
9 Patients 
-1 returned to 
chronic phase 
25 ug/kg 
+ 
hydroxyur
ea 
QOD for 3 
weeks  
-LFT elevations after 7 doses (3 Pt) 
resolved  
-hypocalcemia (4 pt) 
-Sepsis (1 pt) 
-severe bone pain (1 pt) 
-n/v 2 patients 
 
 
 
Baum, M (54) 
1968 
42-83 (mean 
61) 
Various 
-1 
fibrosarcoma 
32 patients 
12- no change 
7 minor 
regression 
1 major 
regression 
(rectal CA) 
25 ug/kg 
over 12 
hrs 
Daily x 8 
11 patients tolerated 8 day course 
-15 4-7 days 
-6 < 4days 
-none-10 
-Anorexia/nausea 9 pts 
-Severe vomiting 5 pts 
-thrombocytopenia 4 pts 
-diarrhea 3 pts 
-CNS symptoms 3 patients 
-thrombophlebitis 3 pts 
SGOT, SGPT, LDH, and AP were 
carefully watched. Occasional patient 
developed a marked rise in LDH in 
absence of other changes in LFTs. 
Only one 
patient got a 
second course 
separated by a 
month 
-stopped with 
vomiting 
 
 
Sewell, I. A. 
(31) 
1966 
30-89 
(mean 56) 
Various 
-1 alveolar 
celled 
-1 
leiomyosarc
oma 
 
26 patients 
-4 pts with 
“quantitative 
remission” 
-6 stable disease 
-another rectal 
cancer 
25 ug/kg 
over 12 
hours 
8 or 10 
doses over 
8 to 14 
days based 
on 
symptoms 
-FEW 
-17 patients with nausea/vomiting 
controlled with phenothiazine (5 pts) 
OR droperidol 
-dizziness and headaches (poorly 
responsive to antihistamines or 
analgesia) 
-seizures in patient with cerebral mets 
- ONLY 2 required cessation of doses 
-NO LFT problems 
-thrombocytopenia in 1 
Not reported 
NB slow 
infusion 
attributed to 
the low side 
effects 
 
 
Ransohoff (41)  
1965 
26-64 
Glioblastom
a 
14 
25 ug/kg 
over 8 
hours 
Daily times 
8 
2 required held drug for 
thrombocytopenia, N/V 
-otherwise limited toxicity 
 
 
3H drug  
crosses 
BBB 


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50 
(8 patients 
with 
improvemen
ts)3 (3+), 2 
(2+), 2 (2+) 
-tx with surgery 
+ radiation 
+mithra 
**** 
Walker (26) 
 
 
1976 
Median age 
53 years 
Anaplastic 
gliomas 
-no 
difference in 
survival 
96 patients at 
NIH 
116 total 
58 got mithra 
-tx with surgery 
+  
randomized to 
mithra +/- 
radiation 
25 
ug/kg/day 
over 6-8 
hours 
Daily times 
21 
-then 6 wks 
later daily 
times 12 
-then 6 wks 
later daily 
times 12 
-83% got 
11 doses 
-median is 
21 doses 
-N/V in 58% of patients 
-mucositis 
-anemia (60%) mild to moderate 
-leukopenia (20%) 
-thrombocytopenia <25,000/µL only 
2 pts.  
-LFTs-mild 30%, moderate-severe 
50% in ALT/AST 
LDH elevation of 18 pts 83 % >400 
units 
 
-hemorrhage into tumor bed in 3 
patients- 2 DEATHS 
-chart summarizing tox 
 
 
Discussi
on of PK 
data at 
the end 
of the 
paper 
-crosses 
BBB but 
should 
not 
(inhibits 
MDR1) 
Kofman 
“metastatic 
ewing;s 
(21) 
1973 
Two 
reported 
21 and 23 yo 
ESFT 
5 patients 
1 durable CR 
25 
ug/kg/day 
as 8-24 
hour 
infusion 
NOTE 
ORAL 
INACTIV
E 
-q3wks 
total of 6-
10 courses 
Thrombocytopenia 
Fever with infusion 
 
 
 
Kofman, s (40) 
1964 
22 
10 
15 
Much 
younger  
3 16 yo 
2 23 yo 
? of 
redundant 
patients 
3 ESFT 
reported 
(2 repeats) 
 
 
 
Not systematically reported  
-thrombocytopenia seen 
 
 
 
Curreri, 
AR(28) 
1960 
36 pts no 
age 
Variety 
A synovial 
5/26 
Embryonal CA 
2 
Chorio 1 
Wilms 1 
Breast 1 
(all at 40 ug/day 
or more) 
25, 30, 35, 
40, 50, 
60,70, 80, 
90 
Daily times 
5 q 5-7 wks 
n/v. thrombocytopenia, hemorrhage, 
hepatic damage (Transaminases, PT, 
Alk Phos) 
Hemorrhage described in some detail 
LFT changes common at doses >50 
mcg/kg/day (increase AP, GOT, 
decrease PT); 2 pts SGOT of 800 U 
 
50 
μg/dose 
 


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51 
Spear (29) 
1963 
58 patients 
(only 29 
with 
performance 
status >50 
Variety of 
adult 
melanoma, 
bronchogeni
c CA, 
hepatpma 
NONE 
50 
ug/kg/dau 
50 x 1 then 
60 
ug/kg/day q 
week x 8 
N/V, severe diarrhea in 3, LFTs 
-thrombocytopenia in 1 patient 
-GI hemorrhage causing death in 1 
N/A 
N/A 
N/A 
Dube (55) 
 
7 pts adults 
Advanced 
metastatic 
cancer 
 
25 to 30 
mcg/kg/da
y as cont. 
infusion 
For 5-13 
days until 
toxicity 
In all pts hematologic and hepatic 
toxicity: decrease WBC and plt, 
increase SGOT 
 
 
 
Package insert 
Pfizer (25) 
1971 
1600 
patients 
Testicular 
CA 
 
25 
ug/kg/day 
over 12 
hours 
daily x 8 
q month 
 
dilute in 
1L of d5 
and give 
over 4-
6hrs 
Q month 
Contraindicated: thrombocytopenia, 
coagulopathy, poor bone marrow, 
follow PLTs, PT and bleeding time 
-hemorrhagic syndrome is dose 
related if 30 ug/kg/day or less 10 or 
fewer bleeds –drug mortality rate is 
1.6% 
IF >30 12% bleeding with mortality 
rate of 5.7% 
COMMON: 
Anorexia, N/V, stomatitis 
LESS COMMON: 
Fever, drowsiness, lethargy, malaise, 
H/A, depression, phlebitis, flushing, 
skin arash 
LABS: 
Thrombocytopenia, leucopenia (6%), 
increased clotting, bleeding time, 
abnormal clot retraction, LFTs, AP, 
LDH, bili 
Renal, Increased BUN, CR and 
proteinuria 
Hypocalcemia, hypophosphatemia, 
hypokalemia 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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11.2 APPENDIX 2: 
 
PERFORMANCE STATUS SCALES/SCORES 
 
 
 


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11.3 APPENDIX 3: STUDY CALENDAR 
Procedurep 
Screening/ 
Baseline** 
Cycle 1 (28 Days) and subsequent cycles 
Post Therapy 
Follow-up 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
 Day 
9 
Day 
10 
Day 
14 
Day 
21 
Day 
28 
At least 30 days 
after last 
mithramycin dosem 
History, PEa 
X 
Xa 
 
 
 
 
 
Xa 
 
 
 
Xa 
Xa 
Xa 
Xa 
Weightaa 
X 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Height (baseline only) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs (BP, HR, RR, temp) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Performance Score 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Histologic Path confirmation 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• 
CBC, differential 
• sodium, potassium, chloride, 
CO2, creatinine, glucose, 
BUN, albumin, calcium, 
magnesium, phosphorus, uric 
acid  LDH and (amylase, 
lipase, baseline only) 
• alk phos, ALT/AST, T 
bilirubin, Direct bilirubin, T 
protein 
X 
 
 Xb 
Xb 
Xb 
Xb 
Xb 
Xb 
 
 Xb 
Xb 
Xb 
Xb 
Xb 
X 
• Plasma sample (1mL)c 
 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
 
• PT/PTT, Thrombin Time, 
Fibrinogend 
Xd 
Xd 
 
 Xd 
Xd 
 Xd 
 
 Xd 
Xd 
 Xd 
 
 
 
 
X 
• HIV, HBV, HCVe 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• Pregnancy Test (urine)f 
Xf 
 Xf 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Procedurep 
Screening/ 
Baseline**
Cycle 1 (28 Days) and subsequent cycles 
Post Therapy 
Follow-up 
Day 
1 
Day 
2 
Da
y 3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
 Day 
9 
Day 
10 
Day 
14 
Day 
21 
Day 
28 
At least 30 
days after last 
mithramycin 
dosem 
• 
 Urinalysis 
 Xff 
 Xff 
 
 
 
 
 
 
 
 
 
 
 
 
 
• Coags:  , PFA-100, VWF 
antigen, VWF activity, Factor 
VIII activity, D-dimer, fibrin 
degredation products, FXIII 
activity 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mithramycin infusion 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Receive oral/IV dexamethasoneo 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 MUGA or echocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12-lead EKGn 
X 
X 
 
 
Xn 
 
 
X 
 
 
 
 
 
 
X 
Radiological Assessments: CT, 
MRI, FDG-PET, Bone scang 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Xg 
X 
Pharmacokineticsh 
 
Xh  
Xh 
 
Xh 
 
 
Xh 
Xh 
(and 
D9) 
 
 
 
 
 
 
Serum Pharmacodynamic Markeri 
 
Xi 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mandatory biopsy for NR0B1 
expressionj 
Xj 
 
 
 
Xj 
 
 
 
 
 
 
 
 
 
 
Adverse Eventsk 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Concomitant Medicationsl 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
**Blood tests within 7 days prior to enrollment; imaging studies within 2 weeks prior to enrollment (UNLESS otherwise stated), histological path evaluation is 
recommended to be completed prior to arrival 
aWeekly during active treatment, and weekly until recovered from toxicity.  


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aaDaily weight recommended for days 2-7 of mithramycin infusion. 
bDaily on days 2 through7, then twice weekly days 9 to 28 of cycles 1 through 3  
c1 mL of plasma will be obtained, filtered through a 0.22 micron filter, frozen and saved by hematology pathology for future reference if needed, prior to receiving 
drug on day 1 of cycle 1 and 2 only. See Error! Reference source not found. for details. 
dCoagulation studies: drawn on days 1, (3, 4 or 5)  and  (7, 8 or 9) or as clinically indicated .  If screening coagulation studies drawn within 1 week of starting drug, 
day 1 can be omitted for cycle 1 only. 
eExcluded by history only.  No laboratory testing of HIV, HBV or HCV is required. 
f required at screening/prior to cycle 1 and prior to each subsequent cycle on females of childbearing potential 
ffrequired at screening/prior to cycle 1 and prior to each subsequent cycle 
gperformed after every EVEN numbered treatment cycle, i.e. cycle 2, cycle 4, etc until off therapy and then every two months until off study or as indicated by 
clinical response criteria see (section 6.2). Imaging studies will be sent to NCI POB for volumetric analysis (see Error! Reference source not found. for 
details). 
hPK samples: prior to and at the completion of the first infusion, then trough and end of infusion samples with day 4 and 7 doses, and 24 and 48 hours after day 7 
dose. Samples will therefore be drawn on days 1, 2, 4, 7, 8, 9 as indicated in Error! Reference source not found. and Error! Reference source not 
found.. 
iA serum sample for potential biomarker analysis will be obtained prior to the first mithramycin injection, and then at the time of each restaging studies. See 
Error! Reference source not found. for details. 
jEligibility and timing for mandatory biopsy is defined in Error! Reference source not found.. 
kSee Error! Reference source not found. to obtain sheet to record adverse events at home 
lSee Error! Reference source not found. to record all medications taken at home 
mPost therapy evaluations will be completed until at least 30 days after the last dose of mithramycin and until resolution of toxicity. 
nAll EKGs should be done prior to infusion. EKG on day 4 to be done on cycle 1 only. 
o2.5 mg dexamethasone/m2 BSA PO or IV starting 12 hours before the first dose of mithramycin, then prior to the first dose, and then twice daily for 24 hours after 
the last dose of mithramycin 
pFor patients who have discontinued mithramycin infusions during any cycle prior to completion of the 7 daily infusions, the required laboratory studies and other 
evaluations can be adjusted by the principal investigator or associate investigator as necessary.


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11.4 APPENDIX 4: PHARMACOKINETIC WORKSHEET FOR MITHRAMYCIN (PHASE I AND PHASE 
II) FOR NURSING STAFF 
DAY 1 
Patient ID 
 
Weight (kg), height (cm), BSA (m2) 
 
Dose level (mg/kg) 
 
Total dose (mg) 
 
Date: 
 
Infusion start time 
 
 
 
Infusion stop time 
 
Pre-dose EKG time: 
 
Signature of person completing 
EKG: 
 
 
Sample # 
Hour 
Target Time  
Actual Time 
 
Signature 
1 
pre dose 
 
 
 
2 
3 h post-start of 
infusion 
 
 
 
3 
Just prior to the 
end of the 6 hour 
infusion 
 
 
 
4 
0.25 h post-
infusion 
 
 
 
5 
0.5 
 
 
 
6 
1 
 
 
 
7 
2 
 
 
 
8 
3 
 
 
 
9 
5 
 
 
 
10 
7 
 
 
 
11* 
9-12  
 
 
 
*If feasible 


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Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of infusion (cannot 
be drawn through the line used to infuse drug, or through another lumen of that line). 
Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant.  Tubes 
should then be placed on crushed ice, and samples should be centrifuged for 15 minutes at approximately 1000 x g at 
0-5 °C within 2 hr after collection.  The plasma should be transferred to separate pre-labeled screw-capped 
polypropylene transfer tubes and stored at -80°C until shipped to the analytical site. 
Patient ID 
 
Weight (kg), height (cm), BSA (m2) 
 
Dose level (mg/kg) 
 
Total dose (mg) 
 
 
Day 2 Date: 
 
Infusion start time: 
 
Stop time: 
 
 
Sample # 
Hours 
Target Time  
Actual Time 
Signature 
1 
pre dose 
 
 
 
2 
End of infusion 
 
 
 
 
Day 4 Date: 
 
Infusion start time: 
 
Stop time: 
 
 
Pre-dose EKG Time: _____________ Signature of person completing EKG: _________________ 
Sample # 
Hours 
Target Time  
Actual Time 
Signature 
1 
pre dose 
 
 
 
2 
End of infusion 
 
 
 
 
Day 7 Date: 
 
Infusion start time: 
 
Stop time: 
 
 
Pre-dose EKG Time: _____________ Signature of person completing EKG: _________________ 
Sample # 
Hours 
Target Time  
Actual Time 
Signature 
1 
pre dose 
 
 
 
2 
End of infusion 
 
 
 
 
Day 8* Date: 
 
Sample # 
Hours 
Target Time  
Actual Time 
Signature 
1 
24 hours after 
day 7 dose 
 
 
 


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2 
24.5 hours after 
day 7 dose 
 
 
 
 
Day 9* Date: 
 
Sample # 
Hours 
Target Time  
Actual Time 
Signature 
*1 
48 hours after 
day 7 dose 
 
 
 
*2 
48.5 hours after 
day 7 dose 
 
 
 
*If feasible 
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of 
infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line). 
Following blood collection, the tubes should be inverted several times to ensure mixing with the 
anticoagulant.  Tubes should then be placed on crushed ice, and samples should be centrifuged for 15 
minutes at approximately 1000 x g at 0-5 °C within 2 hr after collection.  The plasma should be transferred 
to separate pre-labeled screw-capped polypropylene transfer tubes and stored at -80°C.  
Samples will be transported on dry ice to: 
 
Diane Cole 
Pharmacology and Experimental Therapeutics Section 
Pediatric Oncology Branch, NCI 
10 Center Drive, Building 10, room 1-5750. 
Bethesda, MD 201892-1101. 
Phone: 301-496-1757 
E-mail: coled@mail.nih.gov 
 
Please notify Ms. Cole by e-mail or phone prior to transport of the samples. 


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11.5 APPENDIX 5: PHARMACOKINETIC WORKSHEET FOR MITHRAMYCIN (PHASE I AND PHASE 
II) FOR LABORATORY PERSONNEL AND RESEARCH NURSES 
DAY 1 
Patient ID 
 
Weight (kg), height (cm), BSA (m2) 
 
Dose level (mg/kg) 
 
Total dose (mg) 
 
Date: 
 
Infusion start time 
 
 
 
Infusion stop time 
 
 
Sample 
# 
Hour 
Time Specimen 
in Lab 
Time in Centrifuge 
Time in 
Freezer 
Signature 
1 
pre dose 
 
 
 
 
2 
3 h post-start 
of infusion 
 
 
 
 
3 
Just prior to 
the end of the 
6 hour 
infusion 
 
 
 
 
4 
0.25 h post-
infusion 
 
 
 
 
5 
0.5 
 
 
 
 
6 
1 
 
 
 
 
7 
2 
 
 
 
 
8 
3 
 
 
 
 
9 
5 
 
 
 
 
10 
7 
 
 
 
 
11* 
9-12  
 
 
 
 
*If feasible 
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of infusion (cannot 
be drawn through the line used to infuse drug, or through another lumen of that line). 
Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant.  Tubes 
should then be placed on crushed ice, and samples should be centrifuged for 15 minutes at approximately 1000 x g at 


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60 
0-5 °C within 2 hr after collection.  The plasma should be transferred to separate pre-labeled screw-capped 
polypropylene transfer tubes and stored at -80°C until shipped to the analytical site. 
Patient ID 
 
Weight (kg), height (cm), BSA (m2) 
 
Dose level (mg/kg) 
 
Total dose (mg) 
 
 
Day 2 Date: 
 
Infusion start time: 
 
Stop time: 
 
Sample # 
Hours 
Time Specimen in 
Lab 
Time in 
Centrifuge 
Time in 
Freezer 
Signature 
1 
pre dose 
 
 
 
 
2 
End of 
infusion 
 
 
 
 
 
Day 4 Date: 
 
Infusion start time: 
 
Stop time: 
 
Sample 
# 
Hours 
Time Specimen 
in Lab 
Time in 
Centrifuge 
Time in 
Freezer 
Signature 
1 
pre dose 
 
 
 
 
2 
End of 
infusion 
 
 
 
 
 
Day 7 Date: 
 
Infusion start time: 
 
Stop time: 
 
Sample 
# 
Hours 
Time Specimen 
in Lab 
Time in 
Centrifuge 
Time in 
Freezer 
Signature 
1 
pre dose 
 
 
 
 
2 
End of 
infusion 
 
 
 
 
 
Day 8* Date: 
 
Sample 
# 
Hours 
Time Specimen 
in Lab 
Time in 
Centrifuge 
Time in 
Freezer 
Signature 
1 
24 hours after 
day 7 dose 
 
 
 
 


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2 
24.5 hours 
after day 7 
dose 
 
 
 
 
 
Day 9* Date: 
 
Sample # 
Hours 
Time Specimen 
in Lab 
Time in 
Centrifuge 
Time in 
Freezer 
Signature 
*1 
48 hours 
after day 7 
dose 
 
 
 
 
*2 
48.5 hours 
after day 7 
dose 
 
 
 
 
*If feasible 
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of 
infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line). 
Following blood collection, the tubes should be inverted several times to ensure mixing with the 
anticoagulant.  Tubes should then be placed on crushed ice, and samples should be centrifuged for 15 
minutes at approximately 1000 x g at 0-5 °C within 2 hr after collection.  The plasma should be transferred 
to separate pre-labeled screw-capped polypropylene transfer tubes and stored at -80°C.  
Samples will be transported on dry ice to: 
 
Diane Cole 
Pharmacology and Experimental Therapeutics Section 
Pediatric Oncology Branch, NCI 
10 Center Drive, Building 10, room 1-5750. 
Bethesda, MD 201892-1101. 
Phone: 301-496-1757 
E-mail: coled@mail.nih.gov 
 
Please notify Ms. Cole by e-mail or phone prior to transport of the samples.


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11.6 APPENDIX 6: SERUM SAMPLE FOR POTENTIAL BIOMARKER (PHASE I AND PHASE II) 
Materials required:  
• 
Red top glass, no additive, no clot activator with uncoated interior, vacutainer tubes (examples: 
BD366397, BD366442, BD366441). 
• 
Polypropylene screw top freezer tubes, 2 ml (examples: Nalgene LX23633 or Nunc 377267). 
Procedure:  
1) Obtain a peripheral blood sample. Fill red top tube approximately three-fourths full (the tube will 
automatically stop filling when the vacuum is gone). 
2) The tube of whole blood should be allowed to sit for 40 - 50 minutes to allow clotting to complete.   
3) Centrifuge the tube at 3000rpm for 10 minutes.   
4) Using a transfer pipette, remove the serum from the tube and dispense into the freezer tubes provided 
in aliquots of 1 mL. 
5) Label the freezer tube with the study ID number patient ID number, and date. Complete information 
below: 
Sample # 
Patient ID 
number: 
Treatment 
Date of sample 
collection: 
1 
 
Prior to treatment 
 
2 
 
Pre cycle 3 
 
3 
 
Pre cycle 5 
 
4 
 
Pre cycle 7 
 
5 
 
Pre cycle 9 
 
6 
 
Pre cycle__∗ 
 
∗Subsequent samples prior to each restaging cycles. 
6) Freeze at –400C to –800C within 2 hours of phlebotomy.  Freeze the tubes in an upright position. 
7) Store the frozen samples at –400C to –800C.     
8) Deliver samples on dry ice to  
Brigitte Widemann, MD 
NCI, POB 
10 Center Drive, Building 10 CRC 
Room 1-5750, MSC 1101 
Bethesda, MD 20892 
Phone: 301-496-7387 
 
 


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11.7 APPENDIX 7: STUDIES TO BE PERFORMED ON TUMOR TISSUE 
Archival tumor tissue 
a) Archival paraffin embedded tumor tissue for confirmation of EWS/FLI1 fusion transcript to 
determine eligibility for phase II portion of trial, and for evaluation of NR0B1 expression in 
patients with Ewing sarcoma 
Archival paraffin embedded tissue, or if paraffin embedded tissue is not available, 5 consecutive 
unstained slides, should be sent via express carrier to: 
Brigitte Widemann, MD 
NCI, POB 
10 Center Drive, Building 10 CRC 
Room 1-5750, MSC 1101 
Bethesda, MD 20892 
Phone: 301-496-7387 
 
Please notify Dr. Widemann by e-mail (widemanb@mail.nih.gov) prior to shipment.  
Dr. Widemann will submit these samples to the Laboratory of Pathology for testing. 
Tumor tissue from Mandatory tumor biopsies for NR0B1 expression and gene signature 
(Phase II) 
In patients with Ewing sarcoma and EWS-FLI1 fusion transcript ≥ 18 years old, who have 
disease amenable to percutaneous biopsy, and who meet other eligibility criteria for biopsy, a 
biopsy will be obtained prior to treatment and at steady state on treatment during cycle 1. 
Patients who meet these criteria will have to agree to the biopsies in order to being able to 
participate in the trial.  
Tumor Biopsies 
Willingness to undergo tumor biopsies is required for patients with disease amenable to 
percutaneous biopsy on this protocol because modulation of molecular targets in tumor biopsies 
after drug treatment is an important objective of this study.  
Baseline biopsies will be performed following patient enrolling on study. If an initial attempt at 
percutaneous biopsy is unsuccessful, the patient will be given an option to proceed with a 
repeated attempt at percutaneous biopsy. A separate consent form must be signed for each biopsy 
procedure, so patients may choose not to undergo subsequent biopsies. If the baseline biopsy is 
unsuccessful or the patient refuses to undergo subsequent biopsies, no further biopsies will be 
performed, but the patient will remain on study, receive study medication, and other correlative 
studies will be performed. Similarly, patients who miss the first dose of drug will not 
subsequently be biopsied, but will remain on study.  
Timing of tumor biopsies 
For NR0B1 expression and gene expression signature tumor one tumor biopsy each will be 
obtained prior to treatment, and day +4 (+/- 1 day) during cycle 1 at steady state on treatment 
with mithramycin.  
Biopsy procedure 


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No more than 2 tumor biopsy procedures per patient will be performed by Interventional 
Radiology. It is preferred that two core biopsies not less than 18-gauge in diameter and at least 1 
cm in length are obtained. Only percutaneous biopsies will be performed. No biopsy of lung 
metastases will be attempted. 
It is estimated that there will be between 2-5 million cells from each biopsy. If a site is deemed 
appropriate for biopsy with minimal risk to the participant by agreement between the 
investigators and Interventional Radiology, an attempt at biopsy will be made. Determination of 
a disease site amenable to biopsy will be determined on an individual case basis after discussion 
with an interventional radiologist. The biopsy procedure to be used in this protocol is described 
below. Such biopsies can be safely performed as evidenced by literature reports (56) and 
Medical Oncology Branch experience at the Clinical Center. Risks of the procedure include, but 
are not limited to, bleeding, infection, pain, and scarring. We will follow Clinical Center 
Interventional Radiology SOPs for coagulant panel and platelets. 
All biopsies will be by percutaneous approach. No biopsy by an invasive (endoscopic, 
laparoscopic, or surgical) procedure will be performed. Only cutaneous, subcutaneous, or easily 
accessible parenchymal lesion core biopsies will be performed; however, there will be no core 
biopsies of lung lesions. 
The use of imaging to facilitate biopsies will be decided by members of the Interventional 
Radiology team and may include ultrasound, CT scan, or MRI. Should CT scan be needed for 
biopsy, the number of scans for each procedure will be limited to the minimum number needed 
to safely obtain a biopsy. All cases will be carefully reviewed with the interventional radiologists 
at NIH who have extensive experience in performing such procedures. Only if the procedure is 
considered to be low risk will we proceed with tumor biopsy in a given participant. 
Biopsy processing: 
Tissue will be collected and immediately sectioned, a portion will be separated and frozen on dry 
ice for RNA collection for microarray and/or gene signature evaluation of EWS-FLI1 
downstream target expression.  Another portion will be imbedded in paraffin and sectioned.  This 
tissue will be stained using immunohistochemistry for NR0B1 and if tissue remains, proteins in 
the apoptotic pathway will be evaluated including survivin (BIRC5), XIAP, Caspase-8 (CASP8), 
and BCL-XL (BCL2L1). The remaining tissue will be distributed with the goal of establishing 
cell lines or performing basic laboratory investigations. 
Biopsy specimens will be collected by Dr. Brigitte Widemann and distributed to Dr. Helman’s 
laboratory and the Laboratory of Pathology for analysis. 
Brigitte Widemann, MD 
NCI, POB 
10 Center Drive, Building 10 CRC 
Room 1-5750, MSC 1101 
Bethesda, MD 20892 
Phone: 301-496-7387 


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11.8 APPENDIX 8: PLASMA SAMPLES FOR CLINICAL PURPOSE       
 
One mL of plasma will be obtained prior to initiating treatment with mithramycin on cycle 1 and 
prior to cycle 2 for future clinical reference in patients who experience bleeding, if needed. 
Obtain 2 mL of blood, separate plasma by centrifugation, and filter plasma through a 0.22 
micron filter. Freeze the filtered plasma at -800C for future clinical reference if needed. 
Samples will be stored in PETS laboratory freezers. 
 
Plasma sample pre treatment with mithramycin cycle 1: Date obtained:________ 
Plasma sample pre cycle 2 of mithramycin: Date obtained:_________ 
Specific Instructions: 
Obtain blood in 6 ml purple top tube. 
SPIN AT 4000 RPM X 10 Minutes 
OBTAIN FILTERED AND NON-FILTERED SAMPLES 
 
1) Non-Filtered sample – pipette out 1 ml from purple top tube and put into nuc, label as 
non-filtered, and freeze. 
2) Filtered samples –  
a) pipette out greater than 1 ml from purple top tube and put into temporary nuc. 
b) Attach MILLEX GV 0.22 um filter to 5 cc syringe 
c) Attach large bore needle to exposed end of filter 
d) Extract 1 ml from nuc, discard temporary nuc 
e) Transfer from syringe (with filter and needle still attached) to a clean nuc, 
label as FILTERED, and freeze. 
 
Supplies: 
Labels 
3 1.8 ml nucs 
5 ml syringe 
large bore needle 
Millex GV 0.22 um filter 
Pipettes 
Specimen bags 


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11.9 
APPENDIX 9: IMAGING STUDIES FOR VOLUMETRIC ANALYSIS OF TUMOR BURDEN        
As a secondary endpoint, measurable disease will be quantified using volumetric MRI analysis. 
This analysis will be performed at the PETS laboratory. 
Imaging studies will be analyzed by Dr. Eva Dombi. : 
Eva Dombi, MD 
NCI, POB 
10 Center Drive, Building 10 CRC 
Room 1-5750, MSC 1101 
Bethesda, MD 20892 
Phone: 301-451-7023 
 
 
 
 


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11.10  APPENDIX 10: PATIENT LOG FOR REACTIONS/SYMPTOMS        
Patient ID:  ____________________________           Cycle: ___________________ 
 
 
 
Dates of last Mithramycin infusion:      _____________ 
 
Reaction/Symptom  
 
          Start Date    
    End Date  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Contact your study center team or local physician right away if you experience signs of 
bleeding.  This may include, but is not limited to, petechiae (red or purple spots on the skin), 
nosebleeds; bruising; or blood in urine, stool or vomit.   
  
Patient/Parent signature: __________________    Date:______________________ 
 
Reviewed by:_____________________ RN/MD   Date:______________________ 


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11.11 APPENDIX 11: PATIENT LOG FOR CONCOMITANT MEDICATION        
 
Patient ID:____________________________           Cycle: ____________________ 
 
 
 
Dates of last Mithramycin infusion:      _____________ 
 
Medication Taken 
 
 
         
 Start Date   
 
    End Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Contact your study center team or local physician right away if you experience signs of bleeding.  This 
may include, but is not limited to, petechiae (red or purple spots on the skin), nosebleeds; bruising; or 
blood in urine, stool or vomit.   
  
Patient/Parent signature: __________________    Date:______________________ 
 
Reviewed by:_____________________ RN/MD   Date:______________________ 


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11.12 APPENDIX 12:  MONITORING TOXICITIES BETWEEN MITHRAMYCIN CYCLES WITH 
LOCAL PHYSICIAN 
Patient ID: _________________ 
Dates and Doses of last Mithramycin:  ___________________         Cycle: _________ 
Dear Dr. ____________________, 
The patient named above is currently participating on a Phase I-II Mithramycin study conducted at the 
NCI, and requires the following lab tests and evaluations between ____________________  (date) and  
_____________________ (date) 
 
 
o History, physical exam: Once Weekly 
 
o Weight: Once Weekly  
 
o Labs:  (Circle one)  Twice Weekly during Cycles 1-3     or   Once Weekly after Cycle 3. 
 
CBC/Diff 
Chemistry Panel (include sodium, potassium, chloride, CO2, creatinine, glucose, BUN, albumin, 
calcium,  magnesium, LDH, phosphorus, uric acid) 
 
LFT  (include alk phos, ALT/AST, Total bilirubin, Direct bilirubin, Total protein) 
 
Coagulation profiles - ONLY If clinically indicated:* 
 
Please FAX all lab results and medical summaries to:   
 
For any questions, please call: 
NAME OF STUDY CONTACT:   
 
 
 
 
Phone:  
 
For urgent issues, contact the referring investigator at the following phone number:   
(Physician Phone # 
HERE) 
*If the patient experiences any signs of bleeding (≤ Grade 2) including, epistaxis, hematuria, 
hematemesis or blood in the stool, the patient will require immediate comprehensive clinical 
evaluation and coagulation profile (including PT/PTT/fibrinogen, thrombin time, PFA-100, D-
dimer, Ca2+ and ionized calcium)  
Please contact the number above with any clinically significant bleed and support with appropriate 
blood product support. Bleeding that does not stop within 6 hours with appropriate treatment will 
be considered a dose limiting toxicity and further dosing of mithramycin will be discontinued. 
 
_______________________________________       _____________________________________ 
 Physician Signature 
 
 
 
 
Print Name 
Date:    
 
 
 


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