Clinical trial NCT01624090
Conditions: Lung Cancer Esophageal Cancer Mesothelioma Gastrointestinal Neoplasms Breast Cancer
Keywords: Cancer Stem Cell Thoracic Malignancies Mithramycin Treatment Lung Tumors Metastatic Lung Tumors
Sponsor: National Cancer Institute (NCI)
Start date: 2012-09-06; Completion date: 2019-09-27
Enrollment: 16 participants; Allocation: NA; Intervention model: SINGLE_GROUP

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 cancers of the chest, such as lung and esophageal cancer or mesothelioma. Researchers want to see if mithramycin can be used to treat these types of cancer.

Objectives:

\- To see if mithramycin is safe and effective against different chest cancers.

Eligibility:

\- Individuals at least 18 years of age who have lung, esophagus, pleura, or mediastinum cancers.

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.
* Participants will receive mithramycin every day for 7 days, followed by 7 days without treatment. Each 14-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:

Increasing evidence indicates that activation of stem cell gene expression is a common mechanism by which environmental carcinogens mediate initiation and progression of thoracic malignancies. Similar mechanisms appear to contribute to extra-thoracic malignancies that metastasize to the chest. Utilization of pharmacologic agents, which target gene regulatory networks mediating stemness may be novel strategies for treatment of these neoplasms. Recent studies performed in the Thoracic Epigenetics Laboratory, Thoracic and Oncology Surgery Branch (TOSB)/National Cancer Institute (NCI), demonstrate that under exposure conditions potentially achievable in clinical settings, mithramycin diminishes stem cell gene expression and markedly inhibits growth of lung and esophageal cancer and malignant pleural mesothelioma (MPM) cells in vitro and in vivo. These finding add to other recent preclinical studies demonstrating impressive anti-tumor activity of mithramycin in epithelial malignancies and sarcomas that frequently metastasize to the thorax.

Primary Objective:

-To assess clinical response rates of mithramycin administered as 6 hour intravenous infusions in patients with malignancies involving lungs, esophagus, pleura, or mediastinum.

Eligibility:

* Patients with measurable inoperable, histologically confirmed primary lung and esophageal carcinomas, thymic neoplasms, germ cell tumors, malignant pleural mesotheliomas or chest wall sarcomas, as well as patients with gastric, colorectal or renal cancers and sarcomas metastatic to the thorax are eligible.
* Patients with germline single nucleotide polymorphisms (SNPs) in adenosine 5-triphosphate binding cassette subfamily B member 4 (ABCB4), adenosine 5-triphosphate binding cassette subfamily B member 11 (ABCB11), retinal-binding protein (RALBP) or cytochrome P851 (CYP851) that are associated with resistance to mithramycin-induced hepatotoxicity.
* Patients must have had or refused first-line standard therapy for their malignancies.
* Patients must be 18 years or older with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, without evidence of unstable or decompensated myocardial disease. Patients must have adequate pulmonary reserve evidenced by forced expiratory volume 1 (FEV1) and diffusing capacity for carbon monoxide (DLCO) equal to or greater than 30% predicted; oxygen saturation greater than or equal to 92% on room air. Arterial Blood Gas (ABG) will be drawn if clinically indicated.
* Patients must have a platelet count greater than 100,000, an absolute neutrophil count (ANC) equal to or greater than 1500 without transfusion or cytokine support, a normal prothrombin time (PT)/partial thromboplastin time (PTT), and adequate hepatic function as evidenced by a total bilirubin of \<1.5 times upper limits of normal (ULN) and aspartate aminotransferase (AST)/alanine aminotransferase (ALT) less than or equal to 3 x ULN. Serum creatinine less than 3 or equal to 1.6 mg/ml, or creatinine clearance greater than 70 ml/min/1.73m\^2.

Design:

* Simon Optimal Two Stage Design for Phase II Clinical Trials targeting an objective response rate (Response Evaluation Criteria in Solid Tumors (RECIST)) of 30%.
* Patients will be stratified based on location of primary disease (thoracic vs. extra-thoracic).
* Patients will receive 6 hour infusions of mithramycin at 30 -50 mcg/kg every day for 7 days, every 21 days (1 cycle). Three cycles will constitute one course of therapy. Those patients tolerating 30 mcg/kg infusions during the first cycle will receive subsequent cycles of mithramycin at a dose of 50 mcg/kg using the same infusion schedule.
* Following each course of therapy, patients will undergo restaging studies. Patients exhibiting objective response to therapy or stable disease by RECIST criteria will be offered an additional course of therapy.
* Patients exhibiting disease progression will be removed from study.
* Biopsies of index lesions will be obtained at baseline and on day 8 of the second cycle of therapy for analysis of molecular end-points.
* Pharmacokinetics will be assessed during cycle 1 and cycle 2 of the first course of therapy.

Arms: arm 1: Single agent intravenous (IV) mithramycin
Interventions: intervention 1: Mithramycin
Intervention descriptions: intervention 1: 30 mcg/kg intravenous (IV) over 6 hours once daily for 7 days, to be repeated every 21 days (one cycle) until disease progression or unacceptable toxicity

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: Mithramycin for Cancer Stem Cells
Version Date: 09/14/2018
1
Abbreviated Title: Mithramycin for Cancer Stem Cells
CC Protocol #: 12-C-0151 L
Version Date: 09/14/18
NCT Number: NCT01624090
Phase II Evaluation of Mithramycin, an Inhibitor of Cancer Stem Cell Signaling, in 
Patients with Malignancies Involving Lungs, Esophagus, Pleura, or Mediastinum
NCI Principal Investigator:
David S. Schrump, M.D., M.B.A.
Thoracic and Oncology Surgery Branch, NCI, NIH
Building 10, Room 4-3940
10 Center Drive, 
Bethesda, MD 20892-1502
Phone: (240) 760-6239
E-mail: schrumpd@mail.nih.gov
IND Information:
Drug Name:
Mithramycin
IND Number:
115272
Sponsor:
NCI, Center for Cancer Research
Authorized Representative
William Dahut, MD
Manufacturer:
IriSys LLC
Commercial Agents: None


Abbreviated Title: Mithramycin for Cancer Stem Cells
Version Date: 09/14/2018
2
PRÉCIS
Background:
Increasing evidence indicates that activation of stem cell gene expression is a common mechanism 
by which environmental carcinogens mediate initiation and progression of thoracic malignancies. 
Similar mechanisms appear to contribute to extra-thoracic malignancies that metastasize to the 
chest. Utilization of pharmacologic agents, which target gene regulatory networks mediating 
“stemness” may be novel strategies for treatment of these neoplasms. Recent studies performed in 
the Thoracic Epigenetics Laboratory, TOSB/NCI, demonstrate that under exposure conditions 
potentially achievable in clinical settings, mithramycin diminishes stem cell gene expression and 
markedly inhibits growth of lung and esophageal cancer and MPM cells in vitro and in vivo. These 
finding add to other recent preclinical studies demonstrating impressive anti-tumor activity of 
mithramycin in epithelial malignancies and sarcomas that frequently metastasize to the thorax.
Primary Objective:
x
To assess clinical response rates of mithramycin administered as 6 hour intravenous 
infusions in patients with malignancies involving lungs, esophagus, pleura, or 
mediastinum.
Eligibility:
x
Patients with measurable inoperable, histologically confirmed primary lung and 
esophageal carcinomas, thymic neoplasms, germ cell tumors, malignant pleural 
mesotheliomas or chest wall sarcomas, as well as patients with gastric, colorectal or renal 
cancers and sarcomas metastatic to the thorax are eligible.
x
Patients with germline SNPs in ABCB4, ABCB11, RALBP or CYP8B1 that are associated 
with resistance to mithramycin-induced hepatotoxicity.
x
Patients must have had or refused first-line standard therapy for their malignancies.
x
Patients must be 18 years or older with an ECOG performance status of 0 – 2, without 
evidence of unstable or decompensated myocardial disease. Patients must have adequate 
pulmonary reserve evidenced by FEV1 and DLCO equal to or greater than 30% predicted; 
2[\JHQVDWXUDWLRQRQURRPDLUABG will be drawn if clinically indicated.
x
Patients must have a platelet count greater than 100,000, an ANC equal to or greater than 1500 
without transfusion or cytokine support, a normal PT/PTT, and adequate hepatic function as 
evidenced by a total bilirubin of <1.5 x upper limits of normal (ULN) and AST/ALT  3 x 
ULN. Serum creatinine less than or equal to 1.6 mg/ml, or creatinine clearance greater than 
70 ml/min/1.73m2.
Design:
x
Simon Optimal Two Stage Design for Phase II Clinical Trials targeting an objective response 
rate (RECIST) of 30%.
x
Patients will be stratified based on location of primary disease (thoracic vs. extra-thoracic). 
x
Patients will receive 6 hour infusions of mithramycin at 30 -50 mcg/kg every day for 7 days, 
every 21 days (1 cycle). Three cycles will constitute one course of therapy.
Those patients tolerating 30 mcg/kg infusions during the first cycle will receive 
subsequent cycles of mithramycin at a dose of 50 mcg/kg using the same infusion 
schedule.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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3
x
Following each course of therapy, patients will undergo restaging studies. Patients exhibiting 
objective response to therapy or stable disease by RECIST criteria will be offered an 
additional course of therapy.
x
Patients exhibiting disease progression will be removed from study.
x
Biopsies of index lesions will be obtained at baseline and on day 8 of the second cycle of 
therapy for analysis of molecular end-points.
x
Pharmacokinetics will be assessed during cycle 1 and cycle 2 of the first course of therapy.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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TABLE OF CONTENTS
PRÉCIS............................................................................................................................................2 
TABLE OF CONTENTS.................................................................................................................4 
1 INTRODUCTION .....................................................................................................................6 
1.1 Study Objectives .............................................................................................................. 6 
1.2 Background and Rationale:.............................................................................................. 6 
1.3 Mithramycin................................................................................................................... 10 
2 ELIGIBILITY ASSESSMENT AND ENROLLMENT..........................................................14 
2.1 Eligibility Criteria .......................................................................................................... 14 
2.2 Research Eligibility Evaluation...................................................................................... 16 
2.3 Registration Procedures.................................................................................................. 17 
3 STUDY IMPLEMENTATION ...............................................................................................18 
3.1 Study Design .................................................................................................................. 18 
3.2 Drug Administration ...................................................................................................... 18 
3.3 Dosing Delays and Modifications.................................................................................. 19 
3.4 Criteria for Additional Treatment .................................................................................. 21 
3.5 On Study Protocol Evaluation (Appendix 2: Study Calendar) ...................................... 22 
3.6 Criteria for Removal from Protocol Therapy and Off Study Criteria............................ 24 
4 Concomitant medications/measures.........................................................................................24 
4.1 Hematologic and Blood Product Support....................................................................... 24 
4.2 Antiemetic Therapy and transaminitis prophylaxis ....................................................... 24 
4.3 Anti-diarrheal ................................................................................................................. 25 
4.4 Electrolyte Replacement ................................................................................................ 25 
5 Correlative studies / Biospecimen Collection..........................................................................25 
5.1 Pharmacokinetic (PK) Sampling.................................................................................... 25 
5.2 Studies on Tumor tissue................................................................................................. 25 
5.3 Sample Storage, Tracking and Disposition.................................................................... 26 
6 DATA COLLECTION AND EVALUATION........................................................................27 
6.1 Data Collection............................................................................................................... 27 
6.2 Routine Data Collection................................................................................................. 28 
6.3 Exclusions to Routine Data Collection: ......................................................................... 28 


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6.4 Data Sharing Plans ......................................................................................................... 29 
6.5 Response Criteria ........................................................................................................... 29 
6.6 Toxicity Criteria:............................................................................................................ 33 
7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN.......................................................................................................................................34 
7.1 Adverse Event Definitions ............................................................................................. 34 
7.2 NCI-IRB and Clinical Director Reporting ..................................................................... 35 
7.3 IND Sponsor Reporting Criteria .................................................................................... 36 
7.4 Data and Safety Monitoring Plan................................................................................... 37 
8 STATISTICAL CONSIDERATIONS.....................................................................................38 
8.1 Methods of Analysis....................................................................................................... 39 
8.2 Evaluation of Response.................................................................................................. 39 
8.3 Secondary Endpoints:..................................................................................................... 39 
9 COLLABORATIVE AGREEMENTS....................................................................................40 
9.1 Agreement Type............................................................................................................. 40 
10 HUMAN SUBJECTS PROTECTIONS ..................................................................................40 
10.1 Rationale For Subject Selection..................................................................................... 40 
10.2 Participation of Children................................................................................................ 40 
10.3 Participation of Subjects Unable to Give Consent......................................................... 40 
10.4 Evaluation of Benefits and risks/discomforts for all participants .................................. 41 
10.5 Consent Process and Documentation............................................................................. 41 
11 PHARMACEUTICAL INFORMATION................................................................................42 
11.1 Mithramycin (Plicamycin) ............................................................................................. 42 
12 REFERENCES ........................................................................................................................45 
13 TABLES, FIGURES and APPENDICES................................................................................51 
13.1 Tables ............................................................................................................................. 51 
13.2 Figures............................................................................................................................ 54 
13.3 Appendices..................................................................................................................... 68 


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1
INTRODUCTION
1.1
STUDY OBJECTIVES
1.1.1
Primary Objective
x
To determine objective response rates (CR and PR) in patients with malignancies involving 
lungs, esophagus, pleura or mediastinum following mithramycin infusions
1.1.2
Secondary Objective(s)
x
To determine pharmacokinetics and toxicities of mithramycin administered as 6 hour IV 
infusions
x
To ascertain if mithramycin inhibits stem cell gene expression in patients with thoracic 
malignancies
x
To evaluate gene expression, DNA methylation and micro-RNA profiles in pre- and post-
treatment tumor biopsies.
x
To compare gene expression, DNA methylation, and microRNA profiles in patient tumor 
biopsies with treatment response profiles in pre-clinical studies.
x
To examine if mithramycin decreases pluripotent cancer stem cells (side population)
x
To develop methodologies for assessing effects of mithramycin on cancer stem cells, 
hematopoietic stem cells, mesenchymal stem cells, and circulating tumor cells (CTC).
1.2
BACKGROUND AND RATIONALE:
Lung and esophageal cancers, and malignant pleural mesothelioma (MPM) are leading causes of 
cancer-related deaths worldwide 1. In 2012, these malignancies accounted for an estimated 1.8
million deaths globally; in the United States, approximately 160,000 deaths were attributed to
lung cancer, 15,000 deaths were due to esophageal carcinoma and approximately 2,500 deaths
were attributed to MPM 2. Presently, 80% of lung cancers, and 50% of esophageal carcinomas
are directly attributable to cigarette smoke 3,4. Interestingly, whereas tobacco abuse has not been
linked to the pathogenesis of MPM, approximately 70% of patients developing this malignancy
are active or former smokers 5. Currently, more than 1.3 billion people smoke; as such, the global
burden of tobacco-associated thoracic malignancies will continue to increase, with particularly
devastating consequences in developing countries 6.
In addition to being a significant risk factor for major morbidity and mortality in individuals 
undergoing potentially curative resections 7,8, cigarette smoking diminishes responses to chemo-
and radiation therapy, enhances systemic metastases, and decreases survival of patients with
locally advanced or disseminated lung and esophageal cancers and MPM 9-12; the mechanisms
underlying these phenomena have not been fully established. Previously, we reported that under
clinically relevant exposure conditions, cigarette smoke enhances tumorigenicity of lung cancer
cells via polycomb-mediated repression of Dickkopf-1 (Dkk1), which encodes an antagonist
of Wnt signaling 1 3 . In unpublished studies, we observed a similar phenomenon in esophageal
adenocarcinoma cells following cigarette smoke exposure. Additionally, we have observed that
cigarette smoke activates miR-31, targeting Dkk1 as well as several other Wnt antagonists in
lung cancer cells; constitutive expression of this microRNA significantly enhances proliferation
of lung cancer cells in vitro and in vivo 14. In more recent studies 15, we observed that cigarette


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smoke mediates epigenetic repression of miR-487b in lung cancer cells, resulting in over-
expression of polycomb group proteins BMI1 and SUZ12, as Wnt5A, k-ras and C-myc, all of
which have been implicated in modulating stem cell pluripotency 16-19; consistent with these
observations, knock-down of miR-487b increases proliferation and tumorigenicity of lung cancer
cells 1 5. Collectively, these studies suggest that activation of stem cell gene expression may be
a common mechanism by which tobacco components mediate initiation and progression of
thoracic malignancies. As such, utilization of pharmacologic agents, which target gene regulatory
networks mediating “stemness” may be novel strategies for treatment of thoracic malignancies.
Our recent studies, which are summarized below, suggest that under exposure conditions
potentially achievable in clinical settings, mithramycin diminishes stem cell gene expression and
markedly inhibits growth of lung and esophageal cancer and MPM cells in vitro and in vivo.
CSC induces ABCG2 expression in cultured lung and esophageal cancers and MPM cells: A
series of experiments have been performed in the Thoracic Epigenetics Laboratory, TGIB/NCI
to further examine mechanisms by which cigarette smoke enhances “stemness” of thoracic
neoplasms 20. Briefly, Affymetrix microarrays were used to identify gene expression profiles in
cultured lung and esophageal cancer as well as MPM cells mediated by CSC under clinically-
relevant exposure conditions. Interestingly, ABCG2 [also known as breast cancer resistance
protein (BCRP)] was the third to seventh most commonly up-regulated gene in Calu-6, A549, 
EsC1, EsC2, and SB-MES1 and SB-MES2 cells exposed to CSC. Subsequent qRT-PCR 
experiments (Figure 1A upper panel) demonstrated that A549 and EsC2 had relatively high basal 
expression of ABCG2, which was increased 2-4 fold and ~8 fold, respectively, by CSC treatment.
In contrast, Calu-6 and EsC1 exhibited relatively low level basal expression of ABCG2, which was 
augmented approximately 25-30 fold and 6 fold, respectively, by CSC. Interestingly, normal 
aerodigestive tract epithelial cells (SAEC and HET1A) exhibited very low basal levels of ABCG2,
and minimal induction of ABCG2 by CSC. A similar phenomenon was observed for cultured 
normal mesothelial cells (data not shown). Immunofluorescence experiments confirmed that CSC 
exposure increased ABCG2 protein levels in cancer cells (Figure 1A lower panel). CSC-mediated 
induction of ABCG2 was observed across numerous additional lung cancer and MPM lines (data 
not shown). The lack of validated lines precluded further induction experiments using esophageal 
cancer cells.
Clinical Relevance of ABCG2 Activation in Thoracic Malignancies: ABCG2 is a member of the 
ATP binding cassette (ABC) transporters, which functions as a xenobiotic pump in many normal
tissues 21. Its substrates include numerous environmental toxins as well as chemotherapeutic
agents. Several recent studies suggest that ABCG2 is a critical mediator of stem cell homeostasis.
For example, ABCG2 is highly expressed in pluripotent cells, and is an essential upstream
mediator of sonic-hedgehog signaling, which has been implicated in maintenance of stemness
22. Furthermore, ABCG2 binds to heme, thereby diminishing intracellular porphyrin levels,
rendering stem cells resistant to hypoxia 23. Constitutive expression of ABCG2 protects cardiac
stem cells from oxidative stress
2 4, and enhances expansion, while impairing maturation of
hematopoietic progenitor cells 2 5 . Of particular relevance regarding thoracic malignancies are
recent reports demonstrating that increased expression of ABCG2 correlates with chemo-
resistance and stem-like phenotype of lung and esophageal carcinomas 26-30 as well as pleural
mesotheliomas 31-33, and decreased survival of patients with these neoplasms 34-36.
CSC increases side population of cultured cancer cells: Because ABCG2 is highly expressed in 
stem cells, flow cytometry experiments were performed to examine if CSC exposure increased 


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side population (SP), which is believed to be enriched with cancer stem cells 27,29. Representative 
results pertaining to A549 and Calu-6 cells (high and low ABCG2 expressors, respectively) are 
depicted in Figure 1B. Hoechst staining with and without verapamil revealed a SP fraction of 0.23 
% in untreated A549 cells, compared to .95% (a 4-fold increase) in A549 cells exposed to CSC for 
5 days (Figure 1B). The SP fraction in untreated Calu-6 cells (0.06%) was much lower than A549 
cells and increased to 0.98% (a 16 fold increase) following CSC exposure (Figure 1B). Subsequent 
qRT-PCR analysis demonstrated significant increases in ABCG2 expression in SP fractions 
relative to non-SP fractions in A549 and Calu-6 cells (Figure 1C), suggesting that CSC-mediated 
induction of ABCG2 coincides with enhanced pluripotency of cancer cells. This phenomenon was 
less evident in A549 cells, possibly due to the relative levels of ABCG2 induction in these cells 
following CSC exposure.
Role of Aryl Hydrocarbon Receptor and Sp1 in ABCG2 up-regulation by CSC: Purified
carcinogens as well as HDAC inhibitors such as rhomidepsin induce ABCG2 expression in
cancer cells by aryl hydrocarbon receptor (AhR) signaling 37,38. As such, additional experiments 
were performed to examine the mechanisms by which CSC induces ABCG2 expression in lung 
and esophageal cancer cells. qRT-PCR experiments utilizing Calu-6 and EsC1 cells demonstrated 
dose-dependent induction of ABCG2 by benzopyrene, 3-MC as well as TCDD, the magnitude of 
which varied somewhat between the cell lines (Figure 1D; left panel). Interestingly, the AhR 
antagonist, Resveratrol, only partially abrogated CSC-mediated induction of ABCG2 in these cells 
(Figure 1D; right panel Furthermore, knock-down of HDAC6, which is required for activation
and nuclear transport of AhR in response to tobacco carcinogens 39, only modestly diminished 
CSC-mediated induction of ABCG2 in lung and esophageal cancer cells (Figure 2B; upper and 
lower panels). These findings suggested that induction of ABCG2 by CSC was not mediated solely 
by AhR signaling.
In addition to xenobiotic response elements (XRE) that are binding sites for AhR, the ABCG2
promoter contains a number of Specificity protein 1 (Sp1) recognition sites 37 that could mediate 
activation of this gene in response to cigarette smoke. As such, transient transfection experiments 
using ABCG2 promoter-reporter constructs were performed to examine potential roles of AhR and 
Sp1 in mediating ABCG2 activation by CSC. As shown in Figure 2A, serial deletion of XRE as 
well as Sp1 sites markedly diminished ABCG2 promotor activity. Collectively, these data indicated 
that Sp1 contributes significantly to CSC-mediated activation of ABCG2 in cancer cells.
Effects of Mithramycin on CSC-mediated Induction of ABCG2: Additional experiments were
performed to ascertain if mithramycin, which inhibits binding of Sp1 to GC-rich DNA 40 could
repress ABCG2 expression in cancer cells. Briefly, cancer cells were cultured in normal medium 
(NM) with or without CSC in the presence or absence of escalating doses of mithramycin. qRT-
PCR analysis revealed that 24h treatment with mithramycin decreased basal levels of ABCG2 in 
lung and esophageal cancer and MPM cells; furthermore, mithramycin markedly attenuated CSC-
mediated induction of ABCG2 in these cells (representative results pertaining to A549 and Calu-6
are depicted in Figure 2B). Down-regulation of these genes persisted for at least 16 hours 
following removal of mithramycin from culture media (data not shown). Additional analysis 
revealed that mithramycin inhibited basal levels as well as CSC-mediated up-regulation of Sp1
and AhR in these cells. Interestingly, mithramycin also decreased basal as well as CSC-mediated 
expression of Nuclear Factor Erythroid Related Factor 2 (Nrf2), which has been shown recently
to modulate ABCG2 expression
41. Immunofluorescence experiments demonstrated that 
mithramycin decreased ABGC2 expression in cultured cancer cells (Figure 2C; left panel).


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Additional immunoblot experiments revealed that mithramycin mediated dose-dependent 
decreases in Sp1, AhR, and Nrf2 expression, and partially abrogated CSC-mediated increases in 
levels of these transcription factors in A549 and Calu-6 cells (Figure 2C; right panel). Quantitative 
chromatin immunoprecipitation (ChIP) experiments (Figure 2D) demonstrated that CSC induced 
recruitment of Sp1, AhR, and Nrf2 to the ABCG2 promoter; these results were most dramatic for 
Sp1, and were consistent with aforementioned promoter-reporter experiments. Additional ChIP 
analysis revealed that mithramycin diminished CSC-mediated occupancy of these transcription 
factors within the ABCG2 promoter; these effects coincided with appropriate alterations in RNA 
polymerase II (pol II), as well as H3K9Ac and H3K9Me3 (histone activation and repression marks, 
respectively).
Effects of Mithramycin on Proliferation and Tumorigenicity of Cancer Cells: Additional 
experiments were undertaken to examine the effects of mithramycin on proliferation and 
tumorigenicity of cultured thoracic malignancies. Results of this analysis are shown in Figure 3.
MTS experiments demonstrated that 24h mithramycin exposure dramatically inhibited 
proliferation of lung and esophageal cancer as well as MPM cells (Figure 3A). Flow cytometry 
experiments suggested that the growth inhibitory effects of mithramycin were due to cell cycle 
arrest rather than apoptosis (data not shown). Subsequent experiments demonstrated that 
mithramycin administered IP qMWF x 3 weeks mediated significant dose-dependent growth 
inhibition of established subcutaneous tumor xenografts in athymic nude mice (Figure 3B); the 
in-vivo antitumor effects were not associated with appreciable systemic toxicities such as 
decreased activity, skin changes, or significant weight loss. Histopathologic analysis revealed that 
tumors from mithramycin treated mice were less glandular in appearance with somewhat less 
stroma. Furthermore, tumors from mice treated with 2mg/kg mithramycin had 50% fewer mitoses 
relative to control tumors (data not shown). Immunofluorescence experiments confirmed that 
mithramycin decreased ABCG2 expression in tumor xenografts (Figure 3C).
Mechanisms of Mithramycin-Mediated Cytotoxicity: Affymetrix micro-array experiments were 
performed to examine global gene expression profiles in A549 and Calu-6 lung cancer cells ( wt
p53 and p53 null, respectively) cultured in NM with or without mithramycin for 24 hours. 
Mithramycin mediated dramatic dose-dependent alterations in gene expression in these lung 
cancer cells. Highly reproducible results were noted among cell lines and within treatment groups 
(Figure 4A, upper left panel). Using highly stringent criteria of fold change >3 and adjusted 
p<0.01 for drug treatment vs. control, 1582 and 3771 genes were simultaneously modulated in 
A549 and Calu-6 cells following 50 nM and 200 nM mithramycin exposures, respectively (Figure 
4A; lower left panel). 1258 genes were commonly regulated by mithramycin across two cell lines 
and two drug concentrations; the majority of differentially regulated genes were down-regulated 
in both cell lines (Figure 4A right panel). Interestingly, 8 canonical pathways related to stem cell 
signaling were down-regulated by mithramycin in cultured lung cancer cells (Table 1A).
Consistent with these results, mithramycin decreased SP fraction in A549 cells (Figure 4B).
Additional micro-array experiments were performed to examine effects of mithramycin in A549
xenografts (9 each from drug-treated or control mice). Similar to what was observed following in-
vitro drug treatment, mithramycin mediated highly reproducible, dose-dependent alterations in 
gene expression in A549 tumor xenografts (Figure 4C; left panel, and upper right panel). Using 
criteria of fold change >2 and p< 0.05 for drug treatment vs. control, 351 and 1896 genes were 
differentially expressed in xenografts from mice receiving mithramycin at 1 mg/kg and 2 mg/kg, 
respectively, relative to control tumors (Figure 4C; lower right panel). 299 genes were modulated 


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by mithramycin under both doses. ABCG2 was down-regulated > 2 fold in xenografts from mice 
receiving 2mg/kg, but not 1mg/kg mithramycin. All eight of the stem-cell related pathways 
modulated in-vitro by mithramycin, were also targeted in tumor xenografts by systemic drug 
treatment, albeit to a somewhat lesser degree (Table 1B). A similar phenomenon was observed 
regarding the remaining 8 canonical pathways listed in Table 1A. A variety of additional networks 
regulating intracellular signaling, DNA damage response, chromatin remodeling, and 
chromosomal replication were down-regulated in A549 tumor xenografts following mithramycin 
treatment (Figure 5).
1.3
MITHRAMYCIN
1.3.1
Preclinical Studies
Mithramycin, a polyureic acid isolated from streptomyces, was initially evaluated as a
chemotherapeutic agent in cancer patients during the 1960’s and 70’s 42, but was discontinued due
to excessive systemic toxicities
43. Recently there has been renewed interest in clinical 
development of mithramycin and its derivatives because of their ability to specifically inhibit 
binding of Sp1 to GC-rich DNA 40, thereby down-regulating numerous genes mediating stemness,
chemo resistance, invasion and metastasis of cancer cells 20,44-46.. Of particular interest in this regard
are recent studies indicating that currently approved agents such as cyclo-oxygenase inhibitors
markedly enhance mithramycin mediated-inhibition of Sp1 expression/ activity in cancer cells
47. Such combinational strategies could enable reduction of mithramycin doses, and possibly
decrease systemic toxicities in clinical settings.
Recent experiments have been performed to correlate in vivo effects of mithramycin with in vitro
drug exposures in A549 and Calu-6 lung cancer cells. A progressive dose-dependent increase in 
genes commonly regulated in vitro and in vivo by mithramycin was observed (Figure 5). Two to 
ten percent (average 5%) of genes modulated in vitro overlapped with 13-24% (average 18%) of 
genes altered by in vivo mithramycin across various treatment comparisons. 337 genes were 
simultaneously modulated in cultured A549 and Calu-6 cells following 200 nM mithramycin and 
A549 xenografts from mice receiving 2 mg/kg mithramycin IP. Top molecular and cellular 
functions mediated by these 337 genes included stem cell pluripotency, cell cycle progression, 
gene expression, cellular morphology, and death. In all likelihood, the plieotrophic antitumor 
effects of mithramycin are mediated by direct inhibition of Sp1 binding to promoters of master 
genes regulating diverse cellular functions, with subsequent repression of down-stream targets by 
direct as well as indirect mechanisms 48-51 .
Our recent experiments have direct translational implications regarding evaluation of mithramycin 
in patients with thoracic malignancies. Extrapolation of data from previous animal studies 52,53
suggests that tissue mithramycin levels achieved in our xenograft experiments were in the 50-200 
nM range over 24h (Table 2); these exposure conditions, which closely approximated those used
for our in vitro experiments, are potentially achievable using previous mithramycin dosing
schedules in humans
42. As such, gene expression signatures corresponding with treatment 
response in our preclinical studies may be relevant surrogate endpoints in patients receiving 
mithramycin infusions.
Although preclinical data presented thus far have focused primarily on lung and esophageal 
cancers, and malignant pleural mesotheliomas, considerable laboratory efforts have been 
undertaken to evaluate the effects of mithramycin in a variety of other human cancer histologies.


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For example, Sp1 is over-expressed in gastric, colon, breast, pancreas and prostate cancers 54-58)
that frequently metastasize to the thorax. Sp1 over-expression upregulates VEGF and other genes
encoding angiogenic factors in gastric, pancreas, colon and breast cancer cells 48,49,58-63; over-
expression of Sp1 correlates significantly with advanced stage of disease and decreased survival
in patients with gastric or pancreatic carcinomas 54,57,59. Targeted inhibition of Sp1 activity by
mithramycin potently decreases angiogenesis as well as growth of gastric and pancreatic cancer
xenografts 60,64. Collectively these findings, together with data pertaining to the antitumor effects
of mithramycin in sarcomas 65, support evaluation of mithramycin in GI, breast, urologic, and
sarcomatous malignancies that have metastasized to the thorax.
1.3.2
Preclinical toxicology 
In mice, the LD50 was determined to be about 2000 mcg/kg (6000 mcg/m2) of body weight,
which 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)
66. However, mithramycin was found to be 
essentially non-toxic in dogs and monkeys when 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 66). The
most frequently encountered toxicities at higher doses were bleeding, anorexia, vomiting,
elevated liver function tests, electrolyte abnormalities, bone marrow suppression and azotemia 66.
Our preclinical data as well as those reported by Grohar et al 65, indicate that IP doses of 1-2
mg/kg/day three times a week for 3 weeks are well tolerated in mice. In the Grohar study 65,
necropsy demonstrated that mice treated at this dose and schedule had mild hypoalbuminemia,
slightly increased ALT/AST levels with some evidence of liver toxicity described as mild to 
moderate, and minimal thrombocytopenia.
1.3.2.1
Pharmacokinetics
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 67. Preliminary pharmacokinetic parameters of 125 I-plicamycin (mithramycin) in human
plasma using a radioimmunoassay were described 68. 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.
Kennedy et al 52 administered titrated mithramycin by IV or IP routes in mice at doses closely 
approximating 2 mg/kg. Blood, liver, and kidney mithramycin concentrations following IP 
administration were similar if not higher than these observed following IV dosing. Tissue levels 
two hours after administration ranged from ~335 – 425 nM compared to 90 nM in blood (Table 
2). Assuming an elimination half-life of ~10h, tissue levels most likely exceeded 50-100nM for 
>24h.
1.3.3
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 6 8 - 7 2;
however, patient numbers and standard methods of evaluating responses were limited (Appendix 
1). In particular, mithramycin was found to have activity against testicular cancers. In one series of
305 patients with stage III testicular cancer, 10% of patients achieved a complete response, and
an additional 25% showed some evidence of tumor regression 6 6; many of these cases were


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durable responses in patients with widely metastatic disease. In addition, mithramycin exhibited
activity against Ewing sarcoma; two of five patients with EWFT exhibited widespread regression
of metastatic disease following mithramycin treatment, one of whom had a durable complete
response 73. This impressive response rate may be due to the ability of mithramycin to inhibit
expression of EWS-FLI fusion transcript 65. Whereas more than 1,500 patients were treated with
mithramycin, very few (~5) patients with lung cancers and no patients with esophageal
carcinomas or MPM received this drug. Furthermore, only a small number of patients with
breast, ovarian, or GI malignancies were treated with mithramycin. No conclusions can be drawn
regarding clinical activity of mithramycin in these malignancies due to the variability of infusion
schedules and timing of treatment response assessments.
Initial clinical trials suggested that 50 mcg/kg/dose, administered daily times 5 was an optimal
treatment regimen 68. However, Spear et al 74 observed that 70% of 58 patients receiving this
dose and schedule of mithramycin experienced significant toxicity including anorexia, nausea
and vomiting. As a result, the dose of drug used in most subsequent studies was 25 mcg/kg/day
on various schedules. The recommended dose in the Pfizer package insert for adults is 25-30
mcg/kg administered intravenously over 6 hours every 7-10 days of a 28 day cycle. Systemic
toxicities appeared to be dose-related, tending to occur at doses above 30 mcg/kg/dose 66.
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 6 6 ). Unfortunately, there
was no systematic investigation of these toxicities, and it is unclear what levels of supportive care
these patients received. For example, nausea and vomiting occurred quite frequently; however, in
at least two reports, nausea was controlled with phenothiazines or other anti-emetics 43,69,75 .
Hemorrhage was the most profound toxicity observed in patients receiving mithramycin. In
early reports hemorrhage was attributed to thrombocytopenia, and accounted for 3 deaths in 84
patients 42. Subsequent studies revealed that bleeding was dose-related, occurring in 5.4% of 1150
patients treated at 30 mcg/kg/day or more 66. Bleeding tended to occur around day 4 of treatment,
which is atypical of chemotherapy-induced thrombocytopenia. In addition, it appears that platelet
counts remained >20,000/mcL in most of the patients experiencing hemorrhage, suggesting
that bleeding was due to underlying clotting factor deficiencies or dysregulation of coagulation
pathways. 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,
how effective the blood product support was in that era, or what specific clinical and laboratory
abnormalities were seen prior to the hemorrhage. Therefore, the frequency and severity of
hemorrhage from mithramycin administered in the current era are unknown. Nevertheless,
hemorrhage must be considered a rare, but serious potential toxicity of mithramycin therapy.
Additional rare but significant side effects attributable mithramycin include toxic epidermal 
necrolysis 76,77, and possible potentiation of anthracycline-mediated cardiotoxicity 78. The true 
incidences of these toxicities are unknown and are not discussed in the package insert from Pfizer, 
or the previously filed IND for mithramycin.
Mithramycin has also been used for the treatment of malignant hypercalcemia, with 45% of 
patients responding to a single dose of mithramycin in one study 79. However, the bisphosphonate, 
pamidronate, has replaced mithramycin in this setting. For similar reasons, mithramycin was 
investigated for the treatment of Paget’s disease of bone and was found to have good activity in 


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the management of this disease. In one report, 10/10 patients with Paget’s disease reported 
improvements in pain, and an increase in overall activity with treatment 75.
1.3.4
Recent Experience with Mithramycin in Adult Oncology Patients at the NCI
Twelve patients (10 males; 2 females; median age 51y; range 40-72y) with refractory thoracic 
malignancies (six MPMs, 2 esophageal cancers, two lung cancers, 2 synovial sarcomas) received 
6h mithramycin infusions q day x 7, every 28 days. Response rates following two cycles, 
pharmacokinetics, and systemic toxicities (cycle 1) were evaluated. A total of 20 cycles were 
administered to 12 patients; all were evaluable for toxicities. No objective responses were observed 
in 7 patients who were evaluable for treatment responses. No myelosuppression, uncontrollable 
nausea or vomiting was observed. No patients experienced bleeding. The lack of these toxicities, 
which have been associated with mithramycin treatment 66 may be attributable at least in part to 
higher purity of the current drug relative to what had been used previously. 
Eight patients experienced transient, asymptomatic dose-limiting transaminitis following 
25mcg/kg/infusions. This toxicity always occurred on day 4 of the infusion. Percutaneous biopsies
from three individuals revealed apoptotic hepatocellular death (Figure 6A). Four patients (two 
with MPM) had no hepatotoxicity and tolerated dose escalation to 30 mcg/kg/infusion. Peak and 
steady state mithramycin levels were 20 and 8nM, respectively, which were not different in 
patients with or without hepatotoxicity (Figure 6B). DMET genotyping revealed that two SNPs 
(rs2302387 and rs4668115) in genes encoding two related bile transporters (ABCB4 and ABCB11, 
respectively) were significantly associated with LFT elevations in the 12 patients treated with 
mithramycin(Figure 7A and Figure 7B).
Following this observation, we amended the clinical trial to require a priori genotyping of ABCB4 
and ABCB11 in patients with thoracic malignancies. Three additional patients with wild-type 
genotypes at rs2302387 (CC) and rs4668115 (GG) were treated with mithramycin. The first patient 
with stage IV esophageal cancer had a Grade 3 LFT elevation (ALT=347 U/L, AST=314 U/L), 
but this patient also had significant liver disease. We therefore decided to not recruit additional 
patients with liver metastases. The next patient (also with stage IV esophageal cancer) appeared to 
respond to mithramycin without transaminitis (ALT=81 U/L, AST=62 U/L); however, this 
individual died several days later from a spontaneous esophageal perforation that might have been 
secondary to tumor response in a previously radiated field. The final patient experienced LFT 
elevations (ALT=3178 U/L, AST=3695 U/L) that was not explained by genotype. 
To address additional genetic variants that may have contributed to these LFT elevations, we 
conducted additional genotyping analysis using the DMET Plus array. We excluded the patient 
who had liver involvement to avoid confounding the analysis. ABCB4 and ABCB11 genotypes 
retained their significant association with LFT elevation (P<0.040) in spite of an individual with 
grade 4 LFT elevations who was included in this group (Figure 8A-B). Variants in three other 
genes were found to be associated with LFT elevations: rs1143670 in PEPT2 (Figure 8C-D), 
rs12680 in RALBP (Figure 8E-F), and rs6774801 in CYP8B1 (Figure 8G-H). Since PEPT2
genotypes did not demonstrate a genotype dose effect (i.e., the patient with GG had greater ALT 
and AST than those carrying GA), association between LFT elevations and this genotype may be 
confounded by other factors. No relationship between PEPT2 and mithramycin outcomes was 
apparent in the literature. RALBP rs12680 was strongly associated with LFT elevations 
(P=0.0040), 
which 
are 
related 
to 
hepatocellular 
necrosis 
(https://livertox.nlm.nih.gov/Plicamycin.htm). RALBP has previously been associated with 


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promoting hepatic inflammation and LFT elevations in mice 80 and regulates cellular accumulation 
of doxorubicin by a poorly-characterized non-ABC-transport mechanism 81. Mithramycin has a 
conjugated quinone- and semiquinone-rich structure that is similar in structure to doxorubicin, 
suggesting that RALBP may influence cellular accumulation of mithramycin. CYP8B1 is involved 
in the synthesis of cholic acid and thereby influences the composition of the bile pool 82. Since 
ABCB4 and ABCB11 are both involved in bile efflux, it is likely that CYP8B1 and these 
transporters are involved in hepatocellular bile accumulation. Accordingly, we suggest that the 
enrollment criteria of the clinical trial be amended to include the following genotypes: RALBP
rs12680 (GG), CYP8B1 rs6774801 (CC or CA), ABCB4 rs2302387 (CC), and ABCB11 rs4668115 
(GG) (Figure 8I-J). Since RALBP and CYP8B1 variant genotypes are relatively rare (variant allele 
frequency = 13% and 36% respectively), the present genotyping criteria should not preclude 
accrual of patients with thoracic malignancies.
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
2.1
ELIGIBILITY CRITERIA
2.1.1
Inclusion Criteria
2.1.1.1
Patients with measurable (per RECIST see Section 6.5.1.1), inoperable, histologically 
confirmed primary lung and esophageal carcinomas, thymic neoplasms, germ cell tumors, 
malignant pleural mesotheliomas or chest wall sarcomas, as well as patients with gastric, 
colorectal or renal cancers and sarcomas metastatic to the thorax.
2.1.1.2
Histologic confirmation of disease in the Laboratory of Pathology, CCR, NCI, NIH.
2.1.1.3
Disease amenable to biopsy via percutaneous approach or other minimally invasive 
procedures such as thoracoscopy, bronchoscopy, laparoscopy, or GI endoscopy 
2.1.1.4
Age >18.
2.1.1.5
ECOG status 0-2.
2.1.1.6
Patients must have had or refused first-line standard chemotherapy for their inoperable 
malignancies.
2.1.1.7
Patients must have had no chemotherapy, biologic therapy, or radiation therapy for their 
malignancy for at least 30 days prior to treatment. Patients may have received localized 
radiation therapy to non-target lesions provided that the radiotherapy is completed 14 
days prior to commencing therapy, and the patient has recovered from any toxicity. At 
least 3 half-lives must have elapsed since monoclonal antibody treatment. At least six 
weeks must have elapsed between mitomycin C or nitrosourea treatment. 
2.1.1.8
Patients must have adequate organ and marrow function as defined below:
a) Hematologic and Coagulation Parameters:
i.
Peripheral ANC  1500/mm3 
ii.
Platelets  100,000/ mm3 (transfusion independent)
iii.
Hemoglobin  8 g/dL (PRBC transfusions permitted)


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iv.
PT/PTT within normal limits (patient may be eligible for trial if 
abnormality is deemed clinically insignificant and cleared for protocol 
therapy by Hematology Consult service)
b) Hepatic Function
i.
Bilirubin (total) < 1.5 X upper limit of normal (ULN)
ii.
ALT (SGPT)  3.0 X ULN
iii.
Albumin > 2 g/dL
c) Renal Function
i.
Creatinine within normal institutional limits or creatinine clearance 
60 mL/min/1.73 m2
for patients with creatinine levels above 
institutional normal.
ii.
Normal ionized calcium, magnesium and phosphorus (can be on oral 
supplementation)
2.1.1.9
Cardiac Function: Left ventricular ejection fraction (EF) >40% by Echocardiogram,
MUGA, or cardiac MR.
2.1.1.10 Ability of subject to understand, and be willing to sign informed consent
2.1.1.11 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.
2.1.1.12 Patients must be willing to undergo 2 tumor biopsies
2.1.2
Exclusion Criteria
2.1.2.1
Patients with ABCB4, ABCB11, RALBP or CYP8B1 genotypes associated with 
mithramycin-mediated hepatotoxicity.
2.1.2.2
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
2.1.2.3
Patients with cerebral metastases
2.1.2.4
Patients with any of the following pulmonary function abnormalities will be excluded: 
FEV, < 30% predicted; DLCO, < 30% predicted (post-bronchodilator); Oxygen 
VDWXUDWLRQRQURRPDLU$UWHULDOEORRGJDVZLOOEHGUDZQLIFOLQLFDOO\LQGLFDWHG.
2.1.2.5
Patients with evidence of active bleeding, intratumoral hemorrhage or history of bleeding 
diatheses, unless specifically occurring as an isolated incident during reversible 
chemotherapy-induced thrombocytopenia
2.1.2.6
Patients on therapeutic anticoagulation. Note: prophylactic anticoagulation (i.e. 
intralumenal heparin) for venous or arterial access devices is allowed.


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2.1.2.7
Patients who are concurrently receiving or requiring any of the following agents, which
may increase the risk for mithramycin related toxicities, such as hemorrhage:
o
Thrombolytic agents
o
Aspirin or salicylate-containing products, which may increase risk of
hemorrhage
o
Dextran 
o
Dipyridamole 
o
Sulfinpyrazone
o
Valproic acid
o
Clopidogrel
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 mithramycin
2.1.2.11 Patients who in the opinion of the investigator may not be able to comply with the safety 
monitoring requirements of the study.
2.2
RESEARCH ELIGIBILITY EVALUATION
Pre-treatment blood tests should be performed within two weeks and imaging studies within 4 
weeks prior to enrollment on the trial unless otherwise stated. The evaluations required prior to 
starting treatment are listed in table form in Appendix 2: Study Calendar.
x
Complete history and physical examination including assessment of vital signs and ECOG 
status
x
Laboratory
o
ROUTINE LABS: complete blood count, differential, LDH, SGPT (ALT), SGOT (AST), 
alkaline phosphatase, bilirubin (total and direct), BUN, creatinine, amylase, lipase, 
electrolytes, ionized calcium, magnesium, phosphorus, uric acid, albumin and thyroid 
panel.
o
DMET genotyping of PBMC for ABCB4/ABCB11/RALBP/ CYP8B1 SNPs (to be 
performed by the CLIA Molecular Diagnostics Laboratory (CLIA #21D0947274) 
through Dr. William D Figg’s lab under study 06C0014). (May be performed at 
any time prior to enrollment)
o
Urinalysis.
o
Women of child-bearing poWHQWLDOZLOOKDYHDXULQHRUVHUXPȕK&*SUHJQDQF\WHVW Note: 
this test is to be done within 2 days prior to start of treatment.


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o
Coagulation Studies: PT/PTT/Thrombin Time/Fibrinogen, PFA-100, VWF antigen, 
VWF activity, Factor VIII activity, D-dimer, Fibrin degradation products and FXIII 
activity, lupus anticoagulant (LA) test
x
Radiographic Evaluation 
o
Assessment of disease sites by appropriate radiological evaluation. This should include 
a CT scan of chest, abdomen and pelvis, brain MR, PET-CT, and other standard-of-care 
imaging specific for each patient’s malignancy.
x
Cardiac Function Studies
o
Assessment of cardiac function (ejection fraction/shortening fraction) must be performed 
within 4 weeks of enrollment by MUGA, Echocardiogram, or cardiac MR to rule out 
cardiomyopathy.
o
A 12-Lead electrocardiogram (EKG) will also be performed.
x
Pathologic/Tissue Evaluation
o
Histologic confirmation of tumor by the NCI Laboratory of Clinical Pathology, NIH
(may be performed at any time prior to registration).
2.3
REGISTRATION PROCEDURES
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 send via 
encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. 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.
2.3.1
Treatment Assignment Procedures (For registration purposes only)
Cohorts
Number
Name
Description
1
Primary thoracic malignancy
Patients with primary thoracic malignancy
with favorable 
ABCB4/ABCB11/RALBP/CYP8B1 genotypes 
2
Metastatic extra-thoracic 
malignancy
Patients with extra-thoracic malignancy metastatic to lungs, pleura 
or mediastinum with favorable ABCB4/ABCB11/RALBP, CYP8B1
genotypes
3
Thoracic malignancy, 
primary or metastatic -no 
genotyping (Closed)
Subjects enrolled without regard to ABCB4/ABCB11 genotypes
Arms
Number
Name
Description
1
Mithramycin 
Single agent IV mithramycin.


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Arm Assignment
Patients in cohorts 1 and 2 will be directly assigned to arm 1.
The 12 patients in cohort 3 were directly assigned to arm 1. Cohort 3 is now closed.
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN
This study will be conducted as a single site, phase II trial utilizing a Simon Optimal Design. 
Patients will be analyzed in two cohorts on the basis of primary thoracic malignancy vs. extra-
thoracic malignancy metastatic to lungs, pleura or mediastinum. The goal of the study is to 
determine response rates, pharmacokinetics and molecular responses following 6h mithramycin 
infusions as a prelude to combinatorial studies as well as protocols utilizing 24h continuous 
infusions targeting pharmacodynamic endpoints in tumor tissues.
Patients will undergo eligibility evaluation in the Thoracic Surgery Clinic (OP-3). Patients deemed 
eligible for therapy will sign an informed consent and receive mithramycin as described in Section 
3.2. Patients may continue to receive mithramycin as long as they tolerate drug and do not have 
progressive disease (Section 6.4).
Patients on study will be closely monitored for the development of toxicities using regular physical 
examinations, blood pressure monitoring, and laboratory evaluations with particular attention to 
the development of any signs of bleeding. Patients will be admitted to the hospital for the first 
treatment cycle to allow for close monitoring of toxicities. For subsequent cycles, local patients 
may receive infusions in the day hospital; patients traveling from out-of-town will be admitted to 
the hospital. Two weeks (plus 12 or minus 3 days) following each course of therapy (three 21-day 
cycles) patients will undergo appropriate staging studies to determine response to therapy using 
RECIST criteria.
3.2
DRUG ADMINISTRATION
Given our recent findings pertaining to ABCB4, ABCB11, RALBP and CYP8B1 SNPs that 
distinguish patients who did and did not experience mithramycin-mediated hepatotoxicity on this 
trial, the protocol has been amended to accrue only those patients
with favorable 
ABCB4/ABCB11/RALBP/CYP8B1 genotypes. Fourteen of these patients (7 with primary 
thoracic malignancies and 7 with extrathoracic malignancies metastatic to the chest) will receive 
mithramycin infusions during cycle 1 at 30mcg/kg per day. Those patients tolerating 30 mcg/kg 
infusions during the first cycle will receive subsequent cycles of mithramycin at a dose of 50
mcg/kg using the same infusion schedule. Based on the PK data obtained on this trial to date, this 
dose of mithramycin would be expected to produce Cmax of ~40nM and steady state levels of ~ 
20-25nM, concentrations approximating lowest therapeutic doses in preclinical tissue culture 
experiments. Patients exhibiting dose-limiting toxicities during cycle 1 will receive subsequent 
infusions at a dose of 25mcg/kg, with dose reduction guidelines listed in section 3.3. If one or more 
patients in each cohort exhibit response to therapy, an additional 14 patients will be accrued to the 
respective cohort(s) to further define response rates Simon Second Stage). Drug administration in 
the Second Stage will be identical to that in the First stage.


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Mithramycin will be administered intravenously via central line over 6 hours once daily for 7
days to be repeated every 21 days (+7 days/-3 days). Three cycles will constitute one course.
Intravenous hydration will be provided as clinically indicated.
Note: At least 23 hours should elapse between the beginning of one infusion and the beginning of 
the next.
3.3
DOSING DELAYS AND MODIFICATIONS
General guidelines for treatment modifications:
Adverse Events: All adverse events in this trial will be graded using the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 - a complete listing is available at 
the CTEP website: http://ctep.cancer.gov/forms/CTCAEv4.pdf.
Dose adjustments will be made according to the guidelines below, with dose levels defined as 
follows:
Dose Level
Mithramycin Dose
2
50 mcg/kg
1
30 mcg/kg
-1
25 mcg/kg
-2
18 mcg/kg
Summary of dose holding/interruptions and dose de-escalation recommendations for mithramycin
in case of mithramycin -related adverse events (graded according to NCI-CTCAEv4.0)
General Adverse Events
Action
Non-hematological, Grade 1 or 2
(excluding hemorrhage)
x
Continue mithramycin therapy at full dose prescribed. 
x
Apply maximum supportive care recommendations.
x
,ISURORQJHGGXUDWLRQRI*UDGHDGYHUVHHYHQWGD\V
is affecting quality of life, start the next treatment cycle at 
DL -1.
x
If event persists and continues to affect quality of life for 
PRUHGD\V following initial dose reduction, start the 
next treatment cycle at DL -2.
x
If events continue to persist, discontinue drug.
Grade 3 ALT and AST
x
Hold mithramycin therapy until recovery to Grade d 1 (up 
to 21 days of start of cycle). 
x
If ALT and AST resolve to Grade d 1within 21 days, 
maintain current dose level.
x
If ALT and AST do NOT resolve to Grade d 1within 21
days, dose reduce 1 level.
x
If grade 4 ALT or AST occur in a subsequent cycle, dose 
reduce 1 level independent of the date of resolution.
Grade 4 ALT and AST
x
Hold mithramycin therapy until recovery to Grade d 1 (up 
to 21 days of start of cycle). 
x
If ALT and AST resolve to Grade d 1within 21 days, 


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General Adverse Events
Action
reduce 1 level.
x
If grade 4 ALT or AST occur in a subsequent cycle, dose 
reduce 1 level independent of the date of resolution.
Non-hematological, Grade 3 or 4
(excluding ALT, AST, hemorrhage 
and QT prolongation)
x
Apply maximum supportive care recommendations. Hold 
mithramycin therapy until recovery to Grade d 1 (up to 21 
days of start of cycle) and start the next treatment cycle at 
the next lower dose level. Note: if patient’s toxicity 
recovers to Grade d 1 prior to day 7 of treatment cycle, 
dosing for that cycle may resume at one dose level lower. 
Dosing will stop on day 7 (example: grade 3 event occurs 
on day 2 of dosing and resolves by day 5; patient will be 
allowed to receive mithramycin on days 6 and 7 at one 
dose level lower.)
x
If recurrence of adverse event after drug hold/ 
interruptions is observed at the reduced dose, and 
maximum supportive care measures applied, hold drug 
once again until recovery to Grade d 1 (up to 21 days) and 
start the next treatment cycle one dose level lower. Note: 
if patient’s toxicity recovers to Grade d 1 prior to day 7 of 
treatment cycle, dosing for that cycle may resume one 
dose level lower.
x
If symptoms continue to persist at grade 3 or 4, following 
maximal dose reduction, discontinue drug.
QT prolongation, Grade 3 or 4
x
Cardiology consultation will be obtained to determine 
whether any cardiac functional assessment is warranted or 
if therapy should be delayed or discontinued. When 
warranted, ECGs will subsequently be reviewed by the
NCI consulting cardiologist.
x
If therapy is continued, dose reduce 1 level for grade 4 
events
Bleeding Events (hemorrhage)
Grade 1 events
x
Continue mithramycin therapy at full dose prescribed. 
x
Apply maximum supportive care recommendations.
Grade 2 events
x
Apply maximum supportive care recommendations. Hold 
mithramycin therapy until recovery to Grade d 1 (up to 28 
days from start of treatment cycle) and start the next 
treatment cycle at DL -1.
x
If recurrence of adverse event after drug hold/ 
interruptions is observed, and maximum supportive care 
measures applied, hold drug once again until recovery to 
Grade d 1 (up to 21 days from start of treatment cycle) and 
start the next treatment cycle at DL -2.
x
If symptoms continue to persist at any grade, discontinue 
drug.
Grade 3 events
x
Discontinue mithramycin


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General Adverse Events
Action
Grade 4 events
x
Discontinue mithramycin
All grade 2-4 hemorrhage events will initiate full hematologic evaluations as indicated in Section 
3.5.1- Coagulation Studies, as well as formal Hematology consult by Dr. Jay Lozier or one of his 
designates
Hematological Adverse Events
Grades 1 and 2 events
x
Continue mithramycin therapy at full dose prescribed. 
Apply maximum supportive care recommendations.
Grade 3 events
x
Apply supportive care
x
If toxicities resolved to allow retreatment by day 14, 
resume at current dose.
x
If toxicities resolve between days 14-21 to allow 
retreatment (See section 3.4), resume at DL-1.
x
Following dose reduction, if toxicities persist and do not 
allow treatment at day 14, but are sufficiently resolved to 
allow retreatment by day 21, resume at DL-2.
Grade 4 events
x
Apply maximum supportive care recommendations.
x
Hold mithramycin therapy until patient meets hematologic 
criteria for retreatment (See section 3.4) (must be within 
21 days of start of treatment cycle) and start the next 
treatment cycle at DL -1.
x
If recurrence of hematological adverse event after drug 
hold/ interruptions is observed with treatment at DL-1, in 
the setting of maximum supportive care measures applied, 
hold drug once again until patient meets hematologic 
criteria for retreatment (See section 3.4) (must be within 
28 days of start of treatment cycle) and start the next 
treatment cycle at DL -2.
x
If symptoms continue to persist at any grade, discontinue 
drug.
3.4
CRITERIA FOR ADDITIONAL TREATMENT
Patients will be eligible to continue to receive mithramycin for as long as they do not experience 
clinical or radiographic disease progression (See section 6.4) and tolerate mithramycin (as defined 
in Section 3.3).
Patients may receive the next course of mithramycin at the same dose level (or reduced level as 
above) provided the following criteria are met by day 21.
x
patient has stable or responding disease;
x
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meets other laboratory parameters defined in the eligibility section; and
x
has not met any criteria for removal from treatment or off study (Section 3.6)


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3.5
ON STUDY PROTOCOL EVALUATION (APPENDIX 2: STUDY CALENDAR)
3.5.1
Baseline Evaluations
x
Total and fractionated serum bile acid levels within 2 weeks prior to treatment. Note: 
results need not be back prior to commencing treatment.
x
Liver elastography assessment by ultrasound within 2 weeks prior to treatment
3.5.2
Monitoring During Treatment: 
x
Physical exam, to include vital signs, weight, toxicity evaluation and ECOG status within 
4 days of each treatment cycle.
x
Toxicity assessment during cycles 1 and 2: daily during treatment and then weekly. During 
cycle 3 and subsequent cycles, twice weekly during treatment and then weekly.
x
Laboratory evaluations:
o
CBC with differential count within 2 days prior to treatment and daily during drug 
administration (days 1-7 prior to each infusion, active treatment week); then twice 
weekly on off-treatment weeks of cycles 1-2. During subsequent cycles: CBC with 
differential count within 2 days prior to commencing treatment and twice weekly 
during administration prior to infusion, then weekly.
o
Chemistry: sodium, potassium, chloride, CO2, creatinine, glucose, BUN, albumin, 
ionized calcium*, magnesium, alkaline phosphatase, ALT/GPT, AST/GOT, total 
bilirubin, total protein daily within 2 days of commencing treatment and during drug 
administration (days 1-7 prior to each infusion, active treatment week); then twice 
weekly on off-treatment weeks of cycles 1-2. During subsequent cycles chemistries are 
performed weekly.
o
Coagulation Studies: PT/PTT/Thrombin Time, Fibrinogen prior to day 1 and on days 
+4 and +8 of every cycle. 
o
Total and fractionated bile acids within 2 days prior to and on day +7 of every treatment 
cycle and at time of treatment evaluation at the end of the 3rd cycle of therapy.
Note: The patient may have evaluations and lab work obtained through his or her local physician 
between cycles and have the results faxed to the Research Nurse. The Research RN will contact 
the patient as noted above to discuss toxicity.
* Ionized calcium is not part of a standard lab panel and could be omitted if not available or not 
covered by insurance when performed outside NIH. 
x
12-Lead EKG: 
o
Cycles 1 and 2: Days 1, 4, and 7, before infusion within 1 hour of pre infusion PK 
sample to evaluate for QT prolongation potential
Note: although it is preferable to have the EKGs done within 1 hour of PK sample 
collection, EKGs may be done outside of the specified times if unexpected events in 
clinic or floor prevent staff from conducting the study within the specified timeframe. 
EKGs performed outside of this window will NOT be considered protocol deviations.


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o
Cycle 3 and subsequent cycles: Within 1 hour of start of infusion on day 1 and day 7.
x
Tumor Measurements: CT scan of the chest, abdomen and pelvis, brain MR scan as 
clinically indicated, as well as FDG-PET and other standard-of-practice scans relevant to 
each patient’s malignancy, as well as liver elastography by ultrasound will be performed 
two weeks (plus 12 or minus 3 days) after every course of therapy (three cycles).
3.5.3
Research Evaluations
Note: all samples are to be placed on wet ice and transported to the Thoracic Epigenetics Lab 
(TEL) within one hour of draw
x
Cycles 1 and 2 day 1, prior to infusion: 1 mL of plasma will be obtained, filtered through 
a 0.22 micron filter (Kit available in TEL) to be saved for future clinical reference as 
needed
x
Pharmacokinetic (PK) studies - See Appendix 3 for details - PK samples will be processed 
and stored in the TEL and later transported to the NCI, POB, Pharmacology and 
Experimental Therapeutics Section (PETS). Samples will be drawn on days 1, 2, 4, 7, 8, 
and 9. (Note: Patients who do not live locally will not be required to remain in the area for 
the day 9 PK sample.)
x
Baseline (once) and then day 8 of every cycle - Two 10 mL red top tubes and 4 10 mL 
lavender top tubes for the TEL (Note: these are the only specimens collected at each cycle.)
x
Tumor biopsies will be collected once at baseline and again on day 8 of cycle 2 as described 
in Section 5.2.
o
Within two days prior to biopsies: Normal PT/PTT with exception of lupus 
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3.5.4
Follow-up Evaluations:
After completion the last treatment cycle (See section 3.4 for criteria for retreatment), patients will 
be monitored weekly until all clinically significant toxicities resolve to less than or equal to grade 
1 or baseline as follows:
x
Toxicity Evaluation
x
Laboratory evaluation: CBC, Chem20 equivalent including ionized calcium*, PT/PTT 
x
For patients with stable disease or responding to therapy, the following will be done every 
2 months until off study criteria are met:
o
History and physical exam 
o
Laboratory evaluation: CBC, Chem20 equivalent including ionized calcium*, PT/PTT 
o
Tumor Measurements: CT scan of the chest, abdomen and pelvis as well as PET/CT 
and brain MR or CT scan.
Note: The patient may have evaluations and lab work obtained through his or her local physician 
during follow up.
* Ionized calcium is not part of a standard lab panel and could be omitted if not available or not 
covered by insurance when performed outside NIH.


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3.6
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA
3.6.1
Criteria for removal from protocol therapy
x
Patient refusal of further treatments
x
It is deemed in the best interest of the patient.
x
A patient who develops a concurrent serious medical condition that might preclude or 
contraindicate the further administration of mithramycin will be removed from treatment.
x
A patient who becomes pregnant will be immediately taken off therapy. 
x
Excessive toxicity
Note: Patient will be followed until toxicity resolves to baseline or grade 1 or less.
3.6.2
Off-Study criteria
x
Voluntary withdrawal for any reason, or noncompliance with protocol requirements
x
Completion of the follow up period (See section 3.5.4)
x
Progressive disease, unless the patient is being followed for a serious adverse event related 
to the research. The event must resolve to less than or equal to grade 1 or baseline prior to 
removing from the study.
x
If the investigator determines that it is in the best interest of the patient to discontinue 
follow up
x
Death
3.6.3
Off Protocol Therapy and Off Study Procedure
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off 
protocol therapy and when a subject is taken off-study. A Participant Status Update Form from the 
web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed 
and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-
l@mail.nih.gov.
4
CONCOMITANT MEDICATIONS/MEASURES
4.1
HEMATOLOGIC AND BLOOD PRODUCT SUPPORT
Blood product support should be provided to maintain platelets > 20,000 cells/mcl, Hgb > 8.0 
gm/dl and as clinically indicated. Growth factor support is not permitted with exception of 
administration of filgrastim. Filgrastim will not be administered prophylactically but may be 
administered during cycle 1 if a patient experiences neutropenic sepsis, or during subsequent 
cycles if clinically indicated.
4.2
ANTIEMETIC THERAPY AND TRANSAMINITIS PROPHYLAXIS
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 for subsequent cycles. Patients will receive either dexamethasone 8 mg 


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PO or dexamethasone 12mg IV the evening prior to the first dose of mithramycin and then will 
receive dexamethasone 12 mg IV daily through at least the first course of therapy.
4.3
ANTI-DIARRHEAL
Antidiarrheal agents will be prescribed using standard clinical practice guidelines at the preference 
of the investigator.
4.4
ELECTROLYTE REPLACEMENT
Electrolyte replacement will be provided to maintain serum levels within normal limits.
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 and cycle 2 of course 1 (up to 11 
tubes, Refer to details in Appendix 3). In addition, through 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. PK 
analysis will be performed at the NCI, Genitourinary Malignancies Branch (GMB). Samples will 
be stored in the TEL and batched and later transferred to the Figg Lab on dry ice for analysis. 
Please refer to section 5.3.2 for instructions on sample processing.
5.2
STUDIES ON TUMOR TISSUE
Tissue biopsies will be obtained by minimally invasive methods such as CT guided percutaneous,
endoscopic, laparoscopic or video-assisted thoracoscopic techniques at baseline and on day 8 (± 2 
days) of the second cycle. If insufficient tissue was obtained or if there was harm related to the 
first biopsy, a second biopsy will not be obtained. Should a patient experience a dramatic clinical 
response to treatment, additional tumor tissue biopsies may be requested, but are not required. At 
the time of tissue acquisition, immediate on-site cytopathologic analysis will be performed to 
confirm that malignant cells are present in the biopsies, to enhance potential yield and improve 
subsequent evaluation of molecular endpoints. Samples will be placed in saline in an orange top 
tube on wet ice and transported to the Thoracic Epigenetics Lab for further processing. In the 
Thoracic Epigenetics Lab, a portion will be separated and frozen on dry ice for RNA collection 
for microarray and/or gene signature evaluation, using a customized panel of < 100 genes, 
microRNA and DNA methylation sites noted to be modulated in human cancer xenografts in mice 
treated intraperitoneally with mithramycin. If sufficient tissue is available, another portion will be 
imbedded in paraffin for subsequent immunostaining experiments, focusing on expression of 
genes correlating with treatment response in our preclinical experiments. Additional tissue will be 
used to establish cell lines to examine effects of mithramycin on the epigenome and stem cell 
signaling in vitro, in an attempt to correlate these findings with results obtained from analysis of 
tumor biopsies, or to examine SP. All of the analyses, which are predicated on acquisition of 
sufficient biopsy materials, will be performed in the Thoracic Epigenetics Lab/TGIB under 
direction of the PI.
Note: The patient will consent to the optional biopsy at the time of the procedure. If the patient 
refuses the optional biopsy at that time, the refusal will be documented in the medical record and 
in the research record.


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5.3
SAMPLE STORAGE, TRACKING AND DISPOSITION
5.3.1
Thoracic Epigenetics Section, TOSB/NCI
This study will be conducted within the Thoracic Epigenetics Section, TGIB/NCI. Sample 
collection and initial processing will be performed in the Thoracic Epigenetics Laboratory.
Samples will be stored in designated monitored freezers (at least -20°C). 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 Thoracic Epigenetics Section, TOSB/NCI with access limited to the PI or study 
coordinator. Focused gene expression, microRNA, and DNA methylation analysis will be 
performed by nano-string and pyrosequencing techniques in Dr. David Schrump’s laboratory. 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 conclusion of this study, those samples from patients who have not authorized the continued 
use of their specimens for research purposes will be destroyed in accordance with the 
environmental protection laws, regulations and guidelines of the Federal Government and the State 
of Maryland. Any unintentional destruction or loss of the specimens will be reported to the IRB.
Patients who agree to allow continued use of their specimens for research purposes will be enrolled 
on 06-C-0014.
5.3.2
Blood Processing Core (BPC)
Blood Collection
Please e-mail Julie Barnes at Julie.barnes@nih.gov and Paula Carter pcartera@mail.nih.gov at 
least 24 hours before transporting samples (the Friday before is preferred).
For sample pickup, page 102-11964.
For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 
240-760-6190 (main clinical pharmacology lab number).
For questions regarding sample processing, contact Julie Barnes by e-mail or at 240-760-6044.
The samples will be processed, barcoded, and stored in Dr. Figg’s lab until requested by the 
investigator.
Sample Data Collection
All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and 
stored in the LABrador (aka LabSamples) utilized by the BPC. This is a secure program, with 
access to LABrador limited to defined Figg lab personnel, who are issued individual user accounts. 
Installation of LABrador is limited to computers specified by Dr. Figg. These computers all have 
a password restricted login screen. All Figg lab personnel with access to patient information 
annually complete the NIH online Protection of Human Subjects course. 
LABrador creates a unique barcode ID for every sample and sample box, which cannot be traced 
back to patients without LABrador access. The data recorded for each sample includes the patient 


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ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well 
as box and freezer location. Patient demographics associated with the clinical center patient 
number are provided in the system. For each sample, there are notes associated with the processing 
method (delay in sample processing, storage conditions on the ward, etc.).
Sample bar-codes are linked to patient demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol, via registered use of the 
LABrador. It is critical that the sample remains linked to patient information such as race, age, 
dates of diagnosis and death, and histological information about the tumor, in order to correlate 
genotype with these variables
Sample Storage and Destruction
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80qC according 
to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick 
Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be 
accompanied by laboratory staff at all times. 
Access to stored clinical samples is restricted. Samples will be stored until requested by a 
researcher named on the protocol. All requests are monitored and tracked in LABrador. All 
researchers are required to sign a form stating that the samples are only to be used for research 
purposes associated with this trial (as per the IRB approved protocol) and that any unused samples 
must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that 
the samples requested are being used in a manner consistent with IRB approval. 
Following completion of this study, samples will remain in storage as detailed above. Access to 
these samples will only be granted following IRB approval of an additional protocol, granting the 
rights to use the material.
If, at any time, a patient withdraws from the study and does not wish for their existing samples to 
be utilized, the individual must provide a written request. Following receipt of this request, the 
samples will be destroyed (or returned to the patient, if so requested), and reported as such to the 
IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrity 
(i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the IRB.
6
DATA COLLECTION AND EVALUATION 
6.1
DATA COLLECTION
The Principal Investigator will be responsible for the collection, maintenance, and quality control 
of the study data. All clinical data collected for each study subject will be entered into the C3D 
database. Pharmacokinetic and pharmacodynamic research specimens will be entered into 
Labmatrix. The Principal Investigator is responsible for maintaining any source documentation 
related to the study, including any films, tracings, computer discs or tapes. 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. The Sponsor and its monitoring agency will have access to 
C3D and Labmatrix for data monitoring and verification. The investigators will allow study 
monitors and/or the Food and Drug Administration (FDA) to inspect study documents (e.g. consent 


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forms, drug distribution forms, IRB approval) and pertinent hospital or clinical records for 
confirmation of data throughout the study period.
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 breach in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified. 
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
ROUTINE DATA COLLECTION
Following registration, all adverse events will be described in the source documents, reviewed by 
the designated research nurse, and captured in C3D.
x
During the follow up period (more than 30 days following the last treatment), only those 
events that are serious, unexpected, and related to the treatment will be captured in C3D. 
x
All toxicities occurring within 30 days of treatment will be followed until resolution or 
return to baseline.
6.3
EXCLUSIONS TO ROUTINE DATA COLLECTION:
6.3.1
Adverse Events
The following Adverse Events will be captured only in the source documents and will not be 
reported in C3D
x
Laboratory values that do not support the diagnosis of a reportable event
x
All grade 1 events, except for bleeding events, which will all be captured regardless of 
grade.
x
Grade 2 events that are not related to the treatment
x
Grade 1, 2, and 3 lab values drawn outside of the protocol specified time points that are not
assessed as clinically significant by the PI or his designee and are not associated with an 
adverse event
x
Events related to vascular access devices (occlusion, thrombi, hospitalizations for insertion 
or removal)
Note: Events that result in a hospitalization for convenience will not be reported.
6.3.2
Concomitant medications/measures:
x
All concomitant medications and measures will be captured in the source documents. Only 
those medications that the patient is taking at baseline on a routine basis or medications 


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that cause an AE will be captured in C3D (e.g., onetime medications, PRN medications, 
supportive medications, electrolyte replacement and medications given to treat adverse 
events will not be captured in C3D).
6.4
DATA SHARING PLANS
6.4.1
Human Data Sharing Plan
I will share coded, linked human data generated in this research for future research 

in a NIH-funded or approved public repository clinicaltrials.gov

in BTRIS

in publication and/or public presentations
at the time of publication or shortly thereafter
6.4.2
Genomic Data Sharing Plan
The study is not subject to the NIH genomic data sharing policy. The gene and microRNA as well 
as DNA methylation studies performed under this protocol are not genome wide.
6.5
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 83]. 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.
6.5.1
Response Criteria for Radiographic Studies
6.5.1.1
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 83]. 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 
criteria.
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 


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of at least 5 mm. (Note: the appearance of one or more new lesions is also considered 
progressions)
Note: determination of PD will not be made prior to the Day 60 evaluation.
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 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**


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Target 
Lesions
Non-Target 
Lesions
New Lesions
Overall 
Response
Best Overall Response when 
Confirmation is Required*
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.
6.5.2
Confirmatory Measurement/Duration of Response
x
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. 
x
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 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.
6.5.3
Measurable Disease
Measurable lesions are defined as those that can be accurately measured in at least one dimension 
(longest diameter to be recorded) as:
x
By chest x-ray: >20 mm;
x
By CT scan: 
o
Scan slice thickness 5 mm or under as >10 mm with CT scan
o
Scan slice thickness >5 mm: double the slice thickness
x
With calipers on clinical exam: >10 mm.


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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.
6.5.4
Malignant Lymph Nodes 
To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short 
axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 
mm). At baseline and in follow-up, only the short axis will be measured and followed.
6.5.5
Non-Measurable Disease
All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or 
SDWKRORJLFDOO\PSKQRGHVZLWKWRPPVKRUWD[LVDUHFRQVLGHUHGQRQ-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.
6.5.6
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.
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 84.
6.5.7
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. 


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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.
6.5.8
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 84.
6.5.9
Clinical Lesions 
Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules 
and palpable lymph nodes) and t10 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. 
6.5.10 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.
6.5.11 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 
thickness for head and neck lesions.
6.5.12 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.
6.6
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).


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7
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN
7.1
ADVERSE EVENT DEFINITIONS
7.1.1
Adverse Event
An adverse event is any untoward medical occurrence in a human subject, including any abnormal 
sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, 
temporally associated with the subject’s participation in research, whether or not considered 
related to the subject’s participation in the research
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 
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
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:
x
Death
x
A life-threatening adverse drug experience
x
Inpatient hospitalization or prolongation of existing hospitalization
x
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions
x
A congenital anomaly/birth defect.
x
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 


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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. 
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:
x
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
x
Is related or possibly related to participation in the research; AND
x
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 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:
x
All deaths, except deaths due to progressive disease
x
All Protocol Deviations
x
All Unanticipated Problems
x
All non-compliance
Reports must be received within 7 days of PI awareness via iRIS.
7.2.2
NCI-IRB Requirements for PI Reporting Continuing Review
The protocol PI will report to the NCI-IRB: 


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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:
x
All Grade 2 unexpected events that are possibly, probably or definitely related to 
the research; 
x
All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research; 
x
All Grade 5 events regardless of attribution; 
x
All Serious Events regardless of attribution. 
NOTE: Grade 1 events are not required to be reported. 
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.
7.3
IND SPONSOR REPORTING CRITERIA
7.3.1
Expedited Adverse Event Reporting Criteria to the IND Sponsor
During the first 30 days after the subject receives investigational agent/intervention, the
investigator must immediately report to the sponsor, using the mandatory MedWatch form 3500a
or equivalent, 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. For serious adverse events that occur more 
than 30 days after the last administration of investigational agent/intervention, only report those 
that have an attribution of at least possibly related to the agent/intervention.
Required timing for reporting per the above guideline:
x
Deaths (except death due to progressive disease) must be reported via email within 
24 hours. A complete report must be submitted within one business day. 
x
Other serious adverse events as well as deaths due to progressive disease must be 
reported within one business day
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov
and to the CCR PI and study coordinator.
7.3.2
Reporting Pregnancy 
7.3.2.1
Maternal exposure
If a patient becomes pregnant during the course of the study, the study treatment should be 
discontinued immediately and the pregnancy reported to the Sponsor. The potential risk of 
exposure of the fetus to the investigational agent(s) or chemotherapy agents (s) should be 
documented in box B5 of the MedWatch form “Describe Event or Problem”.


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Pregnancy itself is not regarded as an SAE. However, as patients who become pregnant on study 
risk intrauterine exposure of the fetus to agents which may be teratogenic, the CCR is requesting 
that pregnancy should be reported in an expedited manner as Grade 3 “Pregnancy, puerperium 
and perinatal conditions - Other (pregnancy)” under the Pregnancy, puerperium and perinatal 
conditions SOC. 
Congenital abnormalities or birth defects and spontaneous miscarriages should be reported and 
handled as SAEs. Elective abortions without complications should not be handled as AEs. The 
outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy,
normal birth, or congenital abnormality) should be followed up and documented.
If any pregnancy occurs in the course of the study, then the investigator should inform the Sponsor 
within 1 day, i.e., immediately, but no later than 24 hours of when he or she becomes aware of 
it.
The designated Sponsor representative will work with the investigator to ensure that all relevant 
information is provided to the Sponsor within 1 to 5 calendar days for SAEs and within 30 days 
for all other pregnancies.
The same timelines apply when outcome information is available.
7.3.2.2
Paternal exposure
Male patients should refrain from fathering a child or donating sperm during the study and for 60
days after the last dose of Mithramycin.
Pregnancy of the patient’s partner is not considered to be an AE. However, the outcome of all 
pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or 
congenital abnormality) occurring from the date of the first dose until 60 after the last dose should, 
if possible, be followed up and documented.
7.4
DATA AND SAFETY MONITORING PLAN
7.4.1
Principal Investigator/Research Team
The clinical research team will meet regularly when patients are being actively treated on the trial 
to discuss each patient in detail.
All data will be collected in a timely manner and reviewed by the principal investigator or a clinical
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. 
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. 
7.4.2
Sponsor Monitoring Plan
As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 


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SOPs; and human subjects protection. This is done through independent verification of study 
data with source documentation focusing on:
x
Informed consent process
x
Eligibility confirmation
x
Drug administration and accountability
x
Adverse events monitoring
x
Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center.
This trial will be monitored by personnel employed by an CCR contractor. 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.
8
STATISTICAL CONSIDERATIONS
Prior to amendment F, the protocol enrolled 12 evaluable patients, and there were no clinical 
responses, but dose-limiting hepatotoxicity associated with ABCB4 and ABCB11 genotypes was 
identified. Thus, the results from these initial 12 patients will be reported as a separate group, and 
accrual will begin again using the same phase II design as initially proposed, but restricted to 
patients with favorable SNPs, and receiving the modified dose regimen described in amendment 
F. Thus, beginning with amendment F, the primary endpoint of this study will be objective 
response by RECIST criteria v 1.1. Patients will be analyzed in two cohorts (primary thoracic 
malignancy; extra-thoracic malignancy metastatic to the chest). The best response to mithramycin 
will be examined for each cohort. In each cohort, the trial will seek to rule out an unacceptably 
low 5% objective response rate (ORR; p0=0.05), in favor of a higher response rate of 30% 
(p1=0.30). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta = 0.10 
(probability of rejecting a good treatment=0.10), the study will initially enroll 7 evaluable patients 
in each stratum, and if 0 of the 7 have a response, then no further patients will be accrued in that 
cohort. If 1 or more the first 7 in a cohort have a response, then accrual would continue until a total 
of 21 evaluable patients have enrolled in that cohort. As it may take several weeks to determine if 
a patient has experienced a response, a temporary pause in the accrual to the trial may be necessary 
to ensure that enrollment to the second stage is warranted. If there are 1 to 2 responses in 21 
patients, this would be an uninterestingly low response rate, while if there were 3 or more responses 
in 21 patients, then this would be sufficiently interesting to warrant further study of the patients 
from that cohort in later trials. Under the null hypothesis (5% response rate), the probability of 
early termination in each cohort is 70%.
With two cohorts, up to 42 evaluable patients may be required to be accrued, in addition to the 
12 enrolled prior to amendment F. It is expected that 10-15 patients per year may enroll onto this
study. Thus, 3-4 years additional may be needed to enroll 42 additional evaluable patients. To
allow for a small number of inevaluable patients, and to account for the 12 enrolled prior to 
amendment F, the accrual ceiling will be set at 57.
The following table summarizes the two stage design that will be used for each cohort in the 
trial beginning with Amendment F.


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Cumulative 
Number of 
Responses
Decision
Stage 1: Enter 7 patients
0
Terminate the trial for that cohort:
agent ineffective
1 or more
Inconclusive result, continue trial 
(proceed to Stage 2)
Stage 2: Enter 14 additional 
patients
2 or less
Terminate the trial for that cohort:
agent ineffective
3 or more
Terminate the trial: agent effective
8.1
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 85. Toxicity information recorded will include the
type, 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.
8.2
EVALUATION OF RESPONSE 
Any patient who is enrolled and receives at least one full course (4 cycles, 28 doses) 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 
course 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 experience disease progression after the first course
but who did not receive a full course (all 28 doses) will be taken off-treatment and will only be
evaluable for toxicity (not evaluable for response).
8.3
SECONDARY ENDPOINTS:
Pharmacokinetic analysis will be conducted using non-compartmental methods.
For 
pharmacodynamic endpoints, descriptive statistics will be used for each endpoint. Briefly, 


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pharmacokinetics and molecular endpoints in target tissues will be assessed before and after 
mithramycin treatment, with the primary focus being comparison of microarray, qRT-PCR and 
IHC results relative to profiles identified in our preclinical studies. It is anticipated that non-
parametric statistical methods will be used to compare paired results before and after treatment 
(but not between groups), as well as to perform comparisons or correlations among parameters.
As the exact set of comparisons and analyses to be performed will be determined following 
completion of the trials, and will be based on limited numbers of patients, the analyses will be 
considered exploratory and hypothesis generating rather than definitive. The results of these 
descriptive analyses will be used to design further studies.
9
COLLABORATIVE AGREEMENTS 
9.1
AGREEMENT TYPE
N/A
10
HUMAN SUBJECTS PROTECTIONS 
10.1 RATIONALE FOR SUBJECT SELECTION
The patients to be entered on this protocol have advanced malignancies involving lungs, 
esophagus, or pleura and have limited life expectancies. This population was selected because of 
the unknown outcome of this treatment in terms of its effectiveness. The experimental treatment 
has a chance to provide clinical benefit although this is unknown. Subjects of both genders and all 
racial/ethnic groups are eligible. Efforts will be made to extend accrual to a representative 
population, but in this preliminary study, a balance must be struck between patient safety 
considerations and limitations on the number of individuals exposed to potentially toxic and/or 
ineffective treatments on the one hand and the need to explore gender and ethnic aspects of clinical 
research on the other hand. If differences in outcome that correlate with gender or ethnic identity 
are noted, accrual may be expanded, or a follow-up study may be written to investigate these 
differences more fully. One strategy for recruitment may be to distribute an IRB approved protocol 
recruitment letter to General and Oncology physicians and nurses.
10.2 PARTICIPATION OF CHILDREN
It is anticipated that most children with cancer will not have disease that is appropriate for study. 
Because the effects of mithramycin have not been evaluated in children, individuals <18 will be 
excluded from this study until more data are available.
10.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT
Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent 
may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (section 10.4), all subjects will be offered the opportunity to fill in their 
wishes for research and care, and assign a substitute decision maker on the “NIH Advance 
Directive for Health Care and Medical Research Participation” form so that another person can 
make decisions about their medical care in the event that they become incapacitated or cognitively 
impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent 
Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment 
of whether an individual has the capacity to provide consent; assistance in identifying and 
assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint 


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a surrogate. For those subjects that become incapacitated and do not have pre-determined 
substitute decision maker, the procedures described in MAS Policy 87-4 and NIH HRPP SOP 14E 
for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and 
(b) who do not have a legal guardian or durable power of attorney, will be followed.
10.4 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS FOR ALL PARTICIPANTS
There may be some direct benefit to patients who participate in this trial since it is anticipated that 
mithramycin infusion may cause tumor stabilization or some tumor regression. The greatest benefit 
will be the information regarding the feasibility, toxicity and dosing of mithramycin 
administration, as well as the information on changes in cancer stem cell gene expression. The 
risks to the patient participating in this trial are anticipated to be small and are primarily the risks 
associated with administering the two agents and the radiation risk of up to 2 CT guided research 
biopsies. The amount of radiation from the research CT scans is 1.6 rem which is below the 
guidance if 5 rem per year allowed for research subjects by the NIH Radiation Safety Committee.
These potential risks and benefits will be carefully discussed with the patient at the time consent 
is obtained.
10.5 CONSENT PROCESS AND DOCUMENTATION
All patients will be thoroughly screened both via telephone and during clinic evaluation by the 
physician and the research nurse prior to completing the consent. During the initial consultation, 
the patient and family or friends, if present, will be presented with a forthright and detailed 
overview of the treatment option available to them at the NIH. The experimental nature of the 
treatment, its objectives, its theoretical advantages and disadvantages will be presented. The 
Informed Consent document is given to the patient and they are asked to review it, make notes and 
ask questions prior to agreeing to participate in this protocol. The patient is reassured that 
participation on this trial is entirely voluntary and that they can withdraw or decide against 
treatment at any time without adverse consequences. The physician assures the patient that if 
alternative therapy or no therapy at all is preferred, that we will do all that we can to facilitate 
obtaining consultation with the appropriate referral organizations. The Informed Consent 
document may be obtained from the patient by the principal investigator, associate investigators, 
or the medical staff fellow under the supervision of the principal investigator.
Reconsent on this study may be obtained via telephone according to the following procedure: the 
informed consent document will be sent to the subject. An explanation of the study will be 
provided over the telephone after the subject has had the opportunity to read the consent form. The 
subject will sign and date the informed consent. A witness to the subject’s signature will sign and 
date the consent. The original informed consent document will be sent back to the consenting 
investigator who will sign and date the consent form with the date the consent was obtained via 
telephone. A fully executed copy will be returned via mail for the subject’s records. The informed 
consent process will be documented on a progress note by the consenting investigator.
10.5.1 Informed consent of non-English speaking subjects
If there is an unexpected enrollment of a research participant for whom there is no translated extant 
IRB approved consent document, the principal investigator and/or those authorized to obtain 
informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-
2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2). The summary that will be 
used is the English version of the extant IRB approved consent document. Signed copies of both 


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the English version of the consent and the translated short form will be given to the subject or their 
legally authorized representative and the signed original will be filed in the medical record.
Unless the PI is fluent in the prospective subject’s language, an interpreter will be present to 
facilitate the conversation. Preferably someone who is independent of the subject (i.e., not a family 
member) will assist in presenting information and obtaining consent. Whenever possible, 
interpreters will be provided copies of the relevant consent documents well before the consent 
conversation with the subject (24 to 48 hours if possible). 
We request prospective IRB approval of the use of the short form process for non-English speaking 
subjects and will notify the IRB at the time of continuing review of the frequency of the use of the 
Short Form.
11
PHARMACEUTICAL INFORMATION
11.1 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.
11.1.1 Source 
Mithramycin was supplied by IriSys LLC to the NIH Clinical Center Pharmacy, which will be 
providing the mithramycin under an IND held by the CCR.
11.1.2 Toxicity 
The following effects have been observed in previous human administration (possible signs and 
symptoms in parentheses where appropriate)
More frequent:
a) Hypocalcemia (muscle and abdominal cramps)
b) Anorexia (loss of appetite)
c) Diarrhea
d) Stomatitis
e) 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
x
Drowsiness 
x
Fever 
x
Headache
x
Mental depression 
x
Pain, redness, soreness, or swelling at injection site 
x
Unusual tiredness or weakness


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f)
Gastrointestinal bleeding (bloody or black, tarry stools; vomiting of blood)
g) hepatotoxicity (yellow eyes or skin)
h) Epistaxis, hematemesis
i)
Leukopenia (sore throat and fever)—incidence about 6%
j)
Petechial bleeding 
k) Thrombocytopenia 
l)
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. 
11.1.3 Formulation and preparation 
Mithramycin is supplied as 2 mg/vial, freeze dried powder for injection. To prepare the initial 
dilution of 500 mcg (0.5 mg) 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:
Mithramycin dose (mcg) 
Infusion Solution: 0.9% Sodium 
Chloride or 5% Dextrose Injection (mL) 
1000 or above 
1000
500-999
500
250-499
250
All dilutions will be infused over 6 hours. 
11.1.4 Stability and Storage 
Prior to reconstitution, store between -20 and -10 °C (-4 and 14 °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.
11.1.5 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.
11.1.6 Contraindications
The following medications are contraindicated: 
o
Anticoagulants, coumadin- or indandione-derivatives 


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o
Heparin other than heparin flushes
o
Thrombolytic agents
o
Aspirin or salicylate-containing products, which may increase risk of hemorrhage
o
Dextran 
o
Dipyridamole 
o
Sulfinpyrazone 
o
Valproic acid
o
Clopidogrel


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12 REFERENCES
1.
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13 TABLES, FIGURES AND APPENDICES 
13.1 TABLES
13.1.1 Table 1: Genes Differentially Expressed in vitro and in vivo by Mithramycin
Ingenuity Canonical Pathways - in vitro
Differentially expressed genes
Molecular Mechanisms of Cancer
MAPK1, SMAD3, ARHGEF7, LRP6, BMPR2, MAP3K5, 
RBL1, EP300, PTK2, BMPR1A, SOS1, TGFB2, PRKCE, 
AKT3, GSK3B, HIPK2, PRKD1, PRKCA, PMAIP1, 
GNA12, CREBBP, SMAD6, GNAQ, CDK6, TCF3, FZD8, 
BMPR1B, PLCB4, NF1, IRS1, PIK3CB, ARHGEF18, 
CFLAR
TGF-ȕ6LJQDOLQJ
MAPK1, SMAD3, SKI, CREBBP, SMAD6, BMPR2, 
SMURF1, EP300, BMPR1B, BMPR1A, SOS1, TGFB2, 
SMURF2
TR/RXR Activation
SLC2A1, NCOA3, EP300, LDLR, SREBF1, AKT3, STRBP, 
NCOR1, ACACA, PIK3CB, NCOR2, TBL1XR1, RXRA
HER-2 Signaling in Breast Cancer
SOS1, CDK6, PRKCE, AKT3, PIK3CB, MAP3K5, GSK3B, 
PARD3, PRKD1, PRKCA, EGFR
Non-Small Cell Lung Cancer Signaling
STK4, MAPK1, SOS1, CDK6, AKT3, PDPK1, PIK3CB, 
RXRA, PRKCA, EGFR
mTOR Signaling *
MAPKAP1, 
MAPK1, 
PPP2R5C, 
PPP2CA, 
PDPK1, 
RICTOR, EIF4E, IRS1, PRKAA1, EIF3A, PRKCE, AKT3, 
PIK3CB, PPP2R5E, PRKD1, PRKCA
BMP signaling pathway *
BMPR1B, MAPK1, BMPR1A, CREB1, SOS1, CREBBP, 
SMAD6, BMPR2, SMURF1, ATF2
Cyclins and Cell Cycle Regulation
HDAC4, PPP2R5C, PPP2CA, CDK6, TGFB2, GSK3B, 
PPP2R5E
:QWȕ-catenin Signaling *
PPP2R5C, PPP2CA, CSNK1G2, TGFBR3, CREBBP, LRP6, 
GNAQ, CSNK1A1, TCF3, EP300, FZD8, TGFB2, AKT3, 
GSK3B, PPP2R5E, TCF7L2
Human Embryonic Stem Cell Pluripotency *
SMAD3, SMAD6, BMPR2, PDPK1, TCF3, FZD8, 
BMPR1B, BMPR1A, TGFB2, AKT3, PIK3CB, GSK3B, 
TCF7L2
DNA Methylation and Transcriptional Repression 
Signaling
MECP2, ARID4B, SAP18
Role of Oct4 in Mammalian Embryonic Stem Cell 
Pluripotency *
C3orf63, CCNF, JARID2, IGF2BP1, WWP2
Role of NANOG in Mammalian Embryonic Stem 
Cell Pluripotency *
FZD8, BMPR1B, MAPK1, BMPR1A, SOS1, BMPR2, 
AKT3, PIK3CB, GSK3B
Notch Signaling *
MAML2, NUMB, MAML3, JAG1
NF-ț%6LJQDOLQJ
TRAF3, 
TNFRSF1A, 
TGFBR3, 
CREBBP, 
BMPR2, 
MAP4K4, MALT1, EP300, BMPR1B, BMPR1A, IGF1R, 
AKT3, PIK3CB, GSK3B, EGFR
p53 Signaling
PMAIP1, STAG1, AKT3, PIK3CB, GSK3B, HIPK2, EP300


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Ingenuity Canonical Pathways - in vitro
Differentially expressed genes
Molecular Mechanisms of Cancer
MAP2K6, PMAIP1, TCF4, TGFBR1, BMPR2, CRK, RBL1, 
TGFBR2, FZD8, BMPR1B, HIPK2, BRCA1, PRKCA
TGF-ȕ6LJQDOLQJ
MAP2K6, TGFBR2, BMPR1B, TGFBR1, BMPR2
TR/RXR Activation
LDLR, TBL1XR1, RXRA, NCOA3
HER-2 Signaling in Breast Cancer
PARD3, PRKCA
Non-Small Cell Lung Cancer Signaling
STK4, RXRA, PRKCA
mTOR Signaling *
PRKAB2, PPP2R5C, PPP2R5E, PDGFC, PRKCA
BMP signaling pathway *
BMPR1B, BMPR2
Cyclins and Cell Cycle Regulation
HDAC4, PPP2R5C, CCNB2, PPP2R5E
:QWȕ-catenin Signaling *
TGFBR2, FZD8, TCF4, TGFBR1, PPP2R5C, PPP2R5E
Human Embryonic Stem Cell Pluripotency *
TGFBR2, FZD8, BMPR1B, TCF4, TGFBR1, BMPR2, 
PDGFC
DNA Methylation and Transcriptional Repression 
Signaling
SAP30
Role of Oct4 in Mammalian Embryonic Stem Cell 
Pluripotency *
JARID2, BRCA1
Role of NANOG in Mammalian Embryonic Stem 
Cell Pluripotency *
FZD8, BMPR1B, BMPR2
Notch Signaling *
MAML2
NF-ț%6LJQDOLQJ
MAP2K6, TGFBR2, BMPR1B, TGFBR1, BMPR2
p53 Signaling
PMAIP1, TP53INP1, HIPK2, BRCA1
* Stem cell related signaling pathway


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13.1.2 Table 2: Tissue Mithramycin Concentrations in Mice following IV or IP Injection
Assumptions:
1) Specific activity of mithramycin = 147.5 microCi/mg (from reference # 49)
2) Counting efficiency of scintillation counter = 15% (from reference # 49)
3) Specific gravity of C3H mouse liver tissue = 1.086 (from reference # 50 )
4) Specific gravity of C3H mouse kidney tissue = 1.070 (from reference #50)
൬147݉݅ܿݎ݋ܥ݅
݉݃
൰ቆ2.22ݔ10଺ܿ݌݉
݉݅ܿݎ݋ܥ݅
ቇ൬1݉݃
10଺݊݃൰
= 327.5ܿ݌݉
݊݃
(. 15 ݂ܽܿݐ݋ݎ ݂݋ݎ ܿ݋ݑ݊ݐ݅݊݃ ݂݂݁݅ܿ݅݁݊ܿݕ)
1݊ܯ= 1.085݊݃
݉ܮ
30 min post 
injection
2 hrs
post 
injection
Radioactivit
y
Averag
e
Route
Radioactivit
y
Averag
e
Liver (nM)
672
IV
489
713
692
IV
183
336
916
IP
366
814
865
IP
407
387
Kidney (nM)
703
IV
479
1410
1055
IV
261
361
1144
IP
361
996
1075
IP
482
422
Blood (nM)
187
IV
76.6
188
187
IV
104
90.4
325
IP
93.2
305
315
IP
78.5
91.3


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13.2 FIGURES
13.2.1 Figure 1: Effects of CSC on ABCG2 expression in lung and esophageal cancers
A. Upper panel: qRT-PCR analysis demonstrating dose-dependent induction of ABCG2
expression in A549, Calu-6, EsC1 and EsC2 cells as well as cultured normal SAEC and 


Abbreviated Title: Mithramycin for Cancer Stem Cells
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immortalized squamous esophageal epithelia. Basal levels of ABCG2 in cancer cells were higher 
than corresponding normal cells.
Lower panel: Representative immunofluorescence demonstrating increase in ABCG2 expression 
in Calu-6 cells exposed to CSC.
B. Left panel: Representative flow cytometry analysis demonstrating that CSC increases SP 
fraction in Calu-6 and A549 lung cancer cells. The effects of CSC were more pronounced in Calu-
6 relative to A549 cells, exhibiting low vs. high endogenous ABCG2 levels, respectively.
Right panel: Summary of ABCG2 expression for left panel.
C. qRT-PCR analysis of ABCG2 expression in SP and non-SP fractions in A549 and Calu-6 cells.
D. Left panel: qRT-PCR analysis of ABCG2 expression in Calu-6 and EsC1 cells following 
exposure to CSC, or purified carcinogens, which activate AhR signaling.
Right panel: qRT-PCR analysis demonstrating relatively modest inhibition of 1% CSC-mediated 
induction of ABCG2 by the AhR antagonist, resveratrol. 


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Abbreviated Title: Mithramycin for Cancer Stem Cells
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57
13.2.2 Figure 2: Role of AhR, Sp1 and Nrf2 in ABCG2 activation by CSC.
A. Luciferase activity of ABCG2 promoter reporter constructs following transient transfection into 
Calu-6 cells. Relative to full length promoter (ABCG2-1662-LUC), luciferase activities of ABCG2
promoter constructs decreased following serial deletions or mutations of XRE and Sp1 elements.
B. qRT-PCR analysis of ABCG2, AhR, Sp1, and Nrf2 expression in A549 and Calu-6 cells cultured 
in NM with or without mithramycin in the presence or absence of CSC. See text for details.
C. Left panel: Immunofluorescence analysis of ABCG2 expression in A549 cells cultured in the 
presence or absence of mithramycin with or without CSC.
Right panel: Immunoblot analysis of Sp1, AhR, and Nrf2 expression in A549 and Calu-6 cells 
cultured in the presence or absence of mithramycin and/or CSC.
D. Quantitative ChIP analysis of the ABCG2 promoter region in Calu-6 cells cultured in NM with 
or without CSC in the presence or absence of mithramycin. The dose of mithramycin for ChIP was 
optimized in preliminary experiments. See text for details.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Abbreviated Title: Mithramycin for Cancer Stem Cells
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59
13.2.3 Figure 3: Effects of mithramycin in lung and esophageal cancer and malignant pleural 
mesothelioma (MPM) cells.
A. MTS assays depicting effects of 24h mithramycin exposure on proliferation of lung and 
esophageal cancer and MPM cells.
B. Effects of IP mithramycin (1 mg/kg or 2 mg/kg M-W-F x3) on growth of established 
subcutaneous A549, MES1 and MES7 xenografts. Left panel: tumor volumes; middle panel: tumor 
masses; right panel: effects of mithramycin on body mass. 
C. Representative tissue sections from A549 xenografts from control and mithramycin-treated 
mice. Left panel: H&E stains. Right panel: representative immunofluorescence results depicting 
ABCG2 expression in control tumors, and xenografts from mice treated with mithramycin.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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13.2.4 Figure 4: Microarray analysis of mithramycin effects on gene expression cultured A549 
and Calu-6 cells, and A549 xenografts related to respective controls.
Left panel (bottom): Venn diagram demonstrating overlap of genes simultaneously modulated in 
A549 and Calu-6 cells under two in-vitro exposure conditions.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Right panel: Heat map depicting 1258 differentially expressed genes modulated by mithramycin.
A marked dose-dependent alteration of gene expression profiles was observed in these cells 
(triplicate samples).
B. Flow cytometry analysis demonstrating that mithramycin decreases SP in A549 cells.
C. Left panel: Heat map revealing dose-dependent modulation of gene expression by mithramycin
in tumor xenografts.
Right panel: top, PCA demonstrating highly reproducible results of triplicate samples (derived 
from 9 tumors for such conditions); bottom, Venn diagram depicting overlap of genes modulated 
in-vivo under both mithramycin doses.


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13.2.5 Figure 5: Ingenuity Pathway Analysis depicting representative cancer network targeted 
by mithramycin in tumor xenografts following systemic administration of mithramycin 
in athymic nude mice.


Abbreviated Title: Mithramycin for Cancer Stem Cells
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6A 
Mithramycin Induces Hepatotoxicity in Some Cancer 
Patients
Cleaved 
Caspase 3
6B


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Mithramycin Plasma Concentration vs Time 
(20 cycles, 12 pts)
• Peak and steady state concentrations were lower than predicted 
• No significant differences in PK were apparent in patients with or 
without hepatotoxicity
13.2.6 Figure 6: Serum AST and ALT levels and representative liver biopsy from patients with
mithramycin mediated hepatotoxicity.
Transaminitis was associated with apoptotic hepatocyte death (A). Mithramycin levels
during and after 6 hour infusions. No associations were observed between drug levels and 
hepatotoxicity. Drug levels were lower than previously predicted (B).


Abbreviated Title: Mithramycin for Cancer Stem Cells
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65
7A
Mithramycin-Induced Hepatotoxicity Correlates 
with ABCB4 SNP 
7B
ABCB4 and ABCB11 Regulate Bile Acid Flow
Mithramycin
is a member 
of a rare 
class of 
drugs that 
impedes 
sinusoidal 
blood flow 
and therefore 
causes 
central vein 
toxicity.
13.2.7 Figure 7: Associations between ABCB4 and ABCB11 SNPs in mithramycin mediated 
hepatotoxicity in cancer patients


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Abbreviated Title: Mithramycin for Cancer Stem Cells
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67
13.2.8 Figure 8: Results of DMET analysis depicting associations between AST and ALT 
elevations with SNPs in ABCB4, ABCB11, PEPT2, and RALB from patients receiving 
mithramycin
.


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13.3 APPENDICES
13.3.1 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
Donaldson 86
1985
NONE
Kofman 42
“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
“ a surprising 
factor was 
severe 
cumulative 
toxicity
-again 
hemorrhage on 
second course 
with PLT >50
50 ug/kg 
tried
Dutcher 72
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 
79
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 75
1974
47-74 yrs
10 patients 
with Paget’s
10/10 with 
improved pain 
and increased 
activity
(all regimens 
effective)
                
25 ug/kg
Daily x 10 
for cycle 1
Then
A = Daily 
x5 every 
week for a 
month
Vs
B = Q 
week for 14 
months
Vs
-N/V – responded to anti-emetics NO 
dose reductions
-uremia- 7/10 
-thrombocytopenia 1/10 (<150k)
NO hemorrhage-withheld drug if 
PLT<150k
-Isocitrate Dehydrogenase elevation 
1/10
-no evidence of 
cumulative 
hepatotox.


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Author
Year
Patient Age
Disease
Response rate
Dose
Schedule
Toxicity 
In cycle 1
Toxicity 
subsequent 
cycles
MTD?
PK
B1 = twice 
weekly for 
5 months
Johnson, P.R.E
87
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 88
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
Only one 
patient got a 
second course 
separated by a 
month
-stopped with 
vomiting
Sewell, I. A.69
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 71
1965
26-64
Glioblastom
a
(8 patients 
with 
improvemen
ts)3 (3+), 2 
(2+), 2 (2+)
14
-tx with surgery 
+ radiation 
+mithra
25 ug/kg 
over 8 
hours
Daily times 
8
2 required held drug for 
thrombocytopenia, N/V
-otherwise limited toxicity
3H drug 
crosses 
BBB
****
Walker 67
1976
Median age 
53 years
Anaplastic 
gliomas
96 patients at 
NIH
116 total
58 got mithra
25
ug/kg/day 
over 6-8
hours
Daily times 
21
-N/V in 58% of patients
-mucositis
-anemia (60%) mild to moderate
-leukopenia (20%)
Discussi
on of PK 
data at 
the end 


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70
Author
Year
Patient Age
Disease
Response rate
Dose
Schedule
Toxicity 
In cycle 1
Toxicity 
subsequent 
cycles
MTD?
PK
-no
difference in 
survival
-tx with surgery 
+
randomized to 
mithra +/-
radiation
-then 6 wks 
later daily 
times 12
-then 6 wks 
later daily 
times 12
-83% got 
11 doses
-median is 
21 doses
-thrombocytopenia 
-LFTs-mild 30%, moderate50% in 
ALT/AST
-hemorrhage into tumor bed in 3 
patients- 2 DEATHS
-chart summarizing tox
of the 
paper
-crosses 
BBB but 
should 
not 
(inhibits 
MDR1)
Kofman 
“metastatic 
ewing;s
73
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 70
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 68
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
50
ȝJGRVH
Spear 74
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


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Author
Year
Patient Age
Disease
Response rate
Dose
Schedule
Toxicity 
In cycle 1
Toxicity 
subsequent 
cycles
MTD?
PK
Package insert 
Pfizer 66
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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13.3.2 Appendix 2: Study Calendar
 
 
Cycles 1 and 2 
 
Research Eligibility 
Evaluation/Baseline 
Week 1 
Week 2 
 
 
day 1 
day 2 
day 3  
day 4 
day 5 
day 6 
day 7 
 
Mithramycin  
 
x 
x 
x 
x 
x 
x 
x 
 
Histologic path 
confirmation 
x 
 
 
 
 
 
 
 
 
ABCB4 and ABCB11 status 
X 
 
 
 
 
 
 
 
 
Informed consent 
x 
 
 
 
 
 
 
 
 
Medical history 
x 
 
 
 
 
 
 
 
 
Concurrent meds 
x 
 
 
 
 
 
 
 
 
Physical examination 
x 
xi 
 
 
 
 
 
 
 
Performance status 
x 
xi 
 
 
 
 
 
 
 
General labs 
x 
x 
x 
x 
x 
x 
x 
x 
xxb 
Serum bile acids (total and 
fractionated) 
x 
x 
 
 
 
 
 
x 
 
Coagulation labs 
x 
 
 
 
x 
 
 
 
xc 
Pregnancy test 
x 
 
 
 
 
 
 
 
 
Cardiac function tests 
x 
 
 
 
 
 
 
 
 
Imaging studies incl liver 
Elastograpy by ultrasound 
x 
 
 
 
 
 
 
 
 
Adverse events 
 
 
 
Tumor biopsy 
x 
 
 
 
 
 
 
 
 
Research labs 
 
xd 
 
 
 
 
 
 
xc 
PK  
x 
xdf 
xdf 
 
xdf 
 
 
xdf 
xg 
EKG 
x 
xd 
 
 
xd 
 
 
xd 
 
 
Cycles 3 and beyond* 
 
Week 1 
Week 2 
 
day 1 
day 2 
day 3 
day 4 
day 5 
day 6 
day 7 
 
Mithramycin  
x 
x 
x 
x 
x 
x 
x 
 
Histologic path confirmation 
 
 
 
 
 
 
 
 
Informed consent 
 
 
 
 
 
 
 
 
Medical history 
 
 
 
 
 
 
 
 
Concurrent meds 
 
 
 
 
 
 
 
 
Physical examination 
xi 
 
 
 
 
 
 
 


Abbreviated Title: Mithramycin for Cancer Stem Cells
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Cycles 3 and beyond* 
 
Week 1 
Week 2 
 
day 1 
day 2 
day 3 
day 4 
day 5 
day 6 
day 7 
 
Performance status 
xi 
 
 
 
 
 
 
 
General labs 
 
 
 
xh 
 
 
x 
xa 
Coagulation labs 
 
 
 
x 
 
 
 
xc 
Serum bile acids (total and 
fractionated) 
x 
 
 
 
 
 
x 
xe 
(course 1 only) 
Pregnancy test 
 
 
 
 
 
 
 
 
Cardiac function tests 
 
 
 
 
 
 
 
 
Imaging studies including liver 
elastography by ultrasound 
 
 
 
 
 
 
 
xe 
 
Adverse events 
 
Tumor biopsy 
 
 
 
 
 
 
 
 
Research labs 
xd 
 
 
 
 
 
 
xc 
EKG  
xd 
 
 
 
 
 
xd 
 
a - once weekly
b - twice weekly
c - day 8 of cycle (same as day 1 of week 2)
d -prior to dosing
e – two weeks (plus 12 or minus 3 days ) after the end of each course (1 course = 3 cycles)
f - end of infusion
g - 24 and 48 hours after day 7 infusion
h- CBC with differential only
i – within 4 days prior to the start of each cycle
*Patients will continue to receive treatment until criteria for removal from protocol or off-study criteria are met (See section 3.6)


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13.3.3 Appendix 3: SAMPLE Pharmacokinetic and EKG Worksheet for Mithramycin CYCLE 1
TEL research 
sample #
(2 10 mL red top 
tubes & 4 10 mL 
purple top tubes)
Coag reference 
sample #
(obtain kit from
TEL)
PK Sample #
(two 3cc 
lavender top 
tube)
Hour
(pre dose = within 1 
hour prior to 
infusion)
Target Time
Actual Time
EKG 
Time
initials
C1D1
(date)
 
 
 
R1
C 1
PK 1
pre dose
 
 
 
n/a
n/a
PK 2
3 h post-start of 
infusion
 
 
n/a
 
n/a
n/a
PK 3
Just prior to the end 
of the 6 hour infusion
 
 
n/a
 
n/a
n/a
PK 4
0.25 h post-infusion
 
 
n/a
 
n/a
n/a
PK 5
0.5 h post-infusion
 
 
n/a
 
n/a
n/a
PK 6
1 h post-infusion
 
 
n/a
 
n/a
n/a
PK 7
2 h post-infusion
 
 
n/a
 
n/a
n/a
PK 8
3 h post-infusion
 
 
n/a
 
n/a
n/a
PK 9
5 h post-infusion
 
 
n/a
 
n/a
n/a
PK 10
7 h post-infusion
 
 
n/a
 
n/a
n/a
PK 11
9-12 h post-infusion
 
 
n/a
 
C1D2
(date)
 
 
 
n/a
n/a
PK 12
pre dose
 
 
n/a
 
n/a
n/a
PK 13
end of infusion
 
 
n/a
 
C1D4
(date)
 
 
 
n/a
n/a
PK 14
pre dose
 
 
n/a
 
n/a
n/a
PK 15
end of infusion
 
 
n/a
 
C1D7
(date)
 
 
 
n/a
n/a
PK 16
pre dose
 
 
n/a
 
n/a
n/a
PK 17
end of infusion
 
 
n/a
 
C1D8
(date)
 
 
 
R2
n/a
PK 18
24 hrs p/dose #7
  
  
n/a
  
n/a
n/a
PK 19
24.5 hrs/p dose #7
  
  
n/a
  
C1D9
(date)
  
n/a
n/a
PK 20
48 hrs/p dose #7
  
  
n/a
  
n/a
n/a
PK 21
48.5 hrs/p dose #7
  
  
n/a
  


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13.3.4 Appendix 4: SAMPLE Pharmacokinetic and EKG Worksheet for Mithramycin CYCLE 2
TEL research 
samples
(2 10 mL red top 
tubes & 4 10 mL 
purple top tubes)
Coag reference #
(obtain kit from 
TEL)
PK Sample #
(two 3cc 
lavender top 
tube)
Hour
(pre dose = within 1 
hour prior to 
infusion)
Target Time 
Actual Time
EKG 
Time
initials
C2D1
(date)
  
n/a 
C 2
PK 1
pre dose
  
n/a 
n/a 
PK 2
3 h post-start of 
infusion
n/a
  
n/a 
n/a 
PK 3
Just prior to the end 
of the 6 hour infusion
n/a
  
n/a 
n/a 
PK 4
0.25 h post-infusion
n/a
  
n/a 
n/a 
PK 5
0.5 h post-infusion
n/a
  
n/a 
n/a 
PK 6
1 h post-infusion
n/a
  
n/a 
n/a 
PK 7
2 h post-infusion
n/a
  
n/a 
n/a 
PK 8
3 h post-infusion
n/a
  
n/a 
n/a 
PK 9
5 h post-infusion
n/a
  
n/a 
n/a 
PK 10
7 h post-infusion
n/a
  
n/a 
n/a 
PK 11
9-12 h post-infusion
n/a
  
C2D2
(date)
  
n/a
n/a
PK 12
pre dose
  
  
n/a
  
n/a
n/a
PK 13
end of infusion
  
n/a
  
C2D4
(date)
  
n/a
n/a
PK 14
pre dose
  
  
 
  
n/a
n/a
PK 15
end of infusion
  
  
n/a
  
C2D7
(date)
  
n/a
n/a
PK 16
pre dose
  
  
 
  
n/a
n/a
PK 17
end of infusion
  
  
n/a
  
C2D8
(date)
  
R2
n/a
PK 18
24 hrs p/dose #7
  
  
n/a
  
n/a
n/a
PK 19
24.5 hrs/p dose #7
  
  
n/a
  
C2D9
(date)
  


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TEL research 
samples
(2 10 mL red top 
tubes & 4 10 mL 
purple top tubes)
Coag reference #
(obtain kit from 
TEL)
PK Sample #
(two 3cc 
lavender top 
tube)
Hour
(pre dose = within 1 
hour prior to 
infusion)
Target Time 
Actual Time
EKG 
Time
initials
n/a
n/a
PK 20
48 hrs/p dose #7
  
  
n/a
  
n/a
n/a
PK 21
48.5 hrs/p dose #7
  
  
n/a
  
Instructions
PK Studies
Collect 6 mL blood in lavender top tubes. 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. Red top tubes should be kept at room temperature whereas lavender top tubes should be placed immediately 
on crushed ice. Samples should be transferred immediately to the TEL. Lavender top tubes should be centrifuged for 15 minutes at 
approximately 1000 x g at 0-5 °C within 15 minutes of collection. The plasma should be transferred to separate pre-labeled screw-capped 
polypropylene tubes and stored at -80 °C until shipped to the analytical site. Total time from blood draw to freeze down of plasma should be 
less than 1 hour. 
Research Labs
Samples will be collected in 2 red top and 4 lavender top tubes and transported on wet ice immediately to the TEL. Samples in lavender top 
tubes should be centrifuged at 1000 x g at 0-5oC for 15 minutes. Supernatent (plasma) should be transferred to separate pre-labeled screw-
capped polypropylene tubes and stored at -80 °C for subsequent analysis. Pellet (PBMC) should be transferred to separate pre-labeled screw-
capped polypropylene tubes and stored at -80 °C for subsequent analysis. Total time from blood draw to freeze down of plasma should be 
less than 1 hour. Red top tubes should be kept at room temperature for 1 hour. Thereafter, clot should be removed, and then samples centrifuged 
at 1000 x g at 0-5oC for 15 minutes. Supernatent (serum) should be transferred to separate pre-labeled screw-capped polypropylene tubes and 
stored at -80 °C for subsequent analysis. Total time from blood draw to freeze down of serum should be less than 2 hr. 
Coagulation Studies
Samples will transported to the TEL lab on ice within 30 minutes. After centrifugation at 1000 x g at 0-5oC for 15 minutes, supernatant should
be filtered through a 0.22 micron filter, and transferred to separate pre-labeled screw-capped polypropylene tubes and stored at -80 °C for 
subsequent analysis.


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Labeling
Prior to the infusion, the PK /research lab sheet and labels will be placed in the patient's room and a separate set of labels will be given to the 
TEL staff. The labels will list the patient identifiers and either "Cycle1" or "Cycle 2" as appropriate. As the RN (or designee) draws the bloods, 
she will place a label on the tube and write the sample reference # on the label on the tube. When the TEL personnel or fellow processes the 
sample, she will label the storage tube with the label and will transcribe the corresponding sample reference # on the label on the tube.
Tracking
Upon completion of the PK/Research sample draws, the research nurse will make 3 copies of the PK sheet. The original and one copy should 
be placed in the research binder. 2 copies should be given to the TEL personnel. The TEL will retain one copy for their records and will send 
the other copy with the PK samples to Dr. Figg’s lab. Note: Prior to sending the PK sheets to Dr. Figg’s lab, the patient name should be 
crossed out.


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Version Date: 09/14/2018
78
13.3.5 Appendix 5: Sample Patient Log for Reactions/Symptoms
NAME: ____________________________
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 signature: __________________ Date:______________________
Reviewed by:_____________________ RN/MD Date:______________________


Abbreviated Title: Mithramycin for Cancer Stem Cells
Version Date: 09/14/2018
79
13.3.6 Appendix 6: Sample Patient Log for Concomitant Medication
NAME:____________________________
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 signature: __________________ Date:______________________
Reviewed by:_____________________ RN/MD Date:______________________


Abbreviated Title: Mithramycin for Cancer Stem Cells
Version Date: 09/14/2018
80
13.3.7 Appendix 7: SAMPLE Monitoring Toxicities Between Mithramycin Cycles with Local 
Physician
Patient Name: _________________
Dates and Doses of last Mithramycin: ___________________
Cycle: _________
Dear Dr. ____________________,
The patient named above is currently participating on a Phase I-II Mithramycin study at the National Cancer Institute 
at the National Institutes of Health in Bethesda, Maryland, and requires the following lab tests and evaluations between 
____________________ and _____________________: 
(date)
(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, ionized            
calcium**, magnesium, LDH, phosphorus, uric acid)
LFT (include alk phos, ALT/AST, Total bilirubin, Direct bilirubin, Total protein)
Coagulation profiles - ONLY If clinically indicated:*
** only if covered by insurance
Please FAX all lab results and medical summaries to Dr. David Schrump, M.D. or Tricia Kunst, R.N. at 301-451-6934
For any questions, please call 240-760-6239, 240-760-6234, or 240-760-6233 or email tricia.kunst@mail.nih.gov, or
cara.kenney@nih.gov
For urgent issues, contact the Attending Physician, Dr. David Schrump, through the NIH Operator at (301) 496-1211
*,IWKHSDWLHQWH[SHULHQFHVDQ\VLJQVRIEOHHGLQJ*UDGHLQFOXGLQJHSLVWD[LVKHPDWXULDKHPDWHPHVLVRU
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 NIH page operator at 301-496-1211 and ask for the Thoracic Surgery attending on call or
Dr. David Schrump 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 
 NIH/NCI/TGIB  
 

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-0151 
PRINCIPAL INVESTIGATOR: 
David S. Schrump, M.D. 
STUDY TITLE:  
Phase II Evaluation of Mithramycin, an Inhibitor of Cancer Stem Cell Signaling, in 
Patients with Malignancies Involving Lungs, Esophagus, Pleura, or Mediastinum 
Continuing Review Approved by the IRB on 08/06/18 
Amendment Approved by the IRB on 10/15/18 (L) 
Date posted to web:  10/26/18 
Standard 
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.
Why is this study being done?  
The purpose of this study is to test if giving an experimental agent, called mithramycin, can cause 
tumors in lungs, esophagus, pleura, or mediastinum to shrink, and to test the safety of the agent.  
Mithramycin is an experimental drug which was tested as an anti-cancer therapy in the 1960’s 
and was found to have activity (shrink some tumors) against some forms of cancer but was never 
broadly accepted as a treatment due to serious side effects. 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 cancers of the chest, including lung and esophageal cancer and 
mesothelioma as well as sarcomas. 


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-0151 
CONTINUATION: page 2 of 12 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 
 
None of the first twelve patients evaluated on this study responded to therapy; we believe that 
this was due to the fact that we were unable to use higher doses of mithramycin because of 
reversible liver damage in about two-thirds of the patients. However, we were able to determine 
that the liver damage is only associated with certain changes in the genes, ABCB4, ABCB11, 
RALBP and CYP8B1 which code for proteins that are involved in bile flow and drug metabolism 
in the liver. As a result of this, all additional patients enrolled on the study will be those whose 
genes, ABCB4, ABCB11, RALBP and CYP8B1 are of the type that are not associated with liver 
damage from mithramycin. By doing this, we hope to be able to administer higher doses of 
mithramycin more safely. 
Why are you being asked to take part in this study? 
You are being invited to participate in this study because you have cancer and have certain 
genetic characteristics associated with resistance to liver damage caused by mithramycin that is 
not shrinking with known effective therapies.  
How many people will take part in this study?  
As many as 57 patients may participate in this study. If you decide to participate, you will 
undergo a series of tests to make sure you are eligible, including blood tests and scans as 
described below. 
Description of Research Study 
What will happen if you take part in this research study?  
Before you begin the study (work up) 
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. These tests will be performed on a separate 
protocol.  
• 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, MUGA, or cardiac MR - tests that check the function of your 
heart 
• We will also do whatever X-rays, CT scans, or other tests are needed to check 
your 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-0151 
CONTINUATION: page 3 of 12 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 
 
• Review of your tumor tissue to confirm your diagnosis 
• A blood test to determine your ABCB4, ABCB11, RALBP and CYP8B1 gene 
status 
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 21 days. This is called a cycle. Your dose may 
be increased after cycle 1 if there are no unacceptable side effects. 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.  
While you are receiving cycles of mithramycin, you will be watched closely for any adverse 
effects. You will need to have the following procedures and tests: 
• Physical exam every cycle 
• Blood tests daily during mithramycin treatment and twice weekly for the rest of 
the first and second cycles (less often for subsequent cycles)  
• Ultrasound evaluation of the liver prior to the beginning of treatment and each 
time scans and x-rays for tumor evaluation are done (after every 4 cycles) 
Scans and x-rays to evaluate your tumor will be done after every 4 cycles  
Pharmacokinetic Studies (PKs)  
Blood samples will be obtained from you to see how much of the mithramycin is in your blood 
(pharmacokinetics). These samples are about 3 mL (about ½ teaspoon) of blood each and will be 
taken before the drug begins, and periodically, up to 10 additional times during and after 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 2 cycles. A sample may also be obtained 24 and 48 
hours after the day 7 dose. Approximately 60 mL (4-tablespoons) of blood will be drawn for PK 
studies at each of the two cycles.  
Biopsy of Tumor Tissue 
If a small sample of tumor tissue can be safely obtained, you will be required to have a biopsy of 
tumor tissue in order to participate in the study. The biopsy may be done by needle (in which a 
needle is inserted through your skin to collect tumor tissue) or by endoscopy (in which a flexible 
tube with a camera on the end is used to help view the tumor and collect samples from it). 
Biopsies will be done under imaging guidance (CT, MRI, or ultrasound as deemed appropriate by 
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 


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-0151 
CONTINUATION: page 4 of 12 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 
 
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. Sometimes tissue can be obtained safely and comfortably with local anesthesia alone. If 
you require sedation or anesthesia before undergoing a biopsy, you will be informed of the risks 
and you will be asked to sign an additional consent.  
This biopsy will be performed prior to starting treatment and then 8 days (+/- 1) after you have 
started the second cycle of mithramycin 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, but are not 
required for your medical care. Willingness to undergo tumor biopsies is required for taking part 
in this study. No more than two mandatory biopsy procedures will be performed during the study. 
However, if your tumor exhibits dramatic response to mithramycin, additional biopsies may be 
requested, but not required. Such biological information is critical for helping determine how the 
drug is killing cancer cells, and if not, why not. 
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. 
This research study involves exposure to radiation from up to 2 CT scans. This radiation 
exposure is not required for your medical care and is for research purposes only. The amount of 
radiation you will receive in this study is 1.6 rem which is below the guideline of 5 rem (or 0.5 
rem in children) per year allowed for research subjects by the NIH Radiation Safety Committee. 
The average person in the United States receives a radiation exposure of 0.3 rem per year from 
natural sources, such as the sun, outer space, and the earth's air and soil. If you would like more 
information about radiation, please ask the investigator for a copy of the pamphlet, An 
Introduction to Radiation for NIH Research Subjects. 
While there is no direct evidence that the amount of exposure received from participating in this 
study is harmful, there is indirect evidence it may not be completely safe. There may be a very 
slight increase in the risk of cancer.  
Please tell your doctor if you have had any radiation exposure in the past year, either from other 
research studies or from medical tests or care, so we can make sure that you will not receive too 
much radiation. Radiation exposure includes x-rays taken in radiology departments, cardiac 
catheterization, and fluoroscopy as well as nuclear medicine scans in which radioactive materials 
were injected into your body.  
If you are pregnant you will not be permitted to participate in this research study. If you are breast 
feeding and the protocol involves injection of radioactive material you will not be permitted to 
participate. It is best to avoid radiation exposure to unborn or nursing infants since they are more 
sensitive to radiation than adults. 


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-0151 
CONTINUATION: page 5 of 12 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 
 
Follow up and Evaluation of Experimental Regimen  
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. Afterwards, you will visit with 
your local doctor for weekly physical exams and blood work.  
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 should tell your study doctor or nurse at once and 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.  
Effective forms of birth control include: 
• Abstinence  
• intrauterine device (IUD) 
• hormonal [birth control pills, injections, or implants]  
• tubal ligation 
• vasectomy 
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. 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.   
Likely 
• Decreased blood level of calcium 
• Nausea or vomiting 
• Diarrhea 
• Loss of appetite 


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-0151 
CONTINUATION: page 6 of 12 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 
 
• Mouth sores 
• 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) 
• Temporary increase in some liver function studies which should return to normal as soon 
as the drug is stopped 
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 
• Muscle or stomach cramps, possibly due to a low blood level of calcium  
• Abnormal levels of blood elements including phosphorus or potassium. 
• 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. 


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-0151 
CONTINUATION: page 7 of 12 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 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). A separate informed 
consent will be obtained should you need a blood transfusion. 
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 unlikely that this treatment will cure your cancer. Because there is not 
much information about the effect of mithramycin on the type of cancer that you have, 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. 
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 (such as hormone 
therapy and/or systemic chemotherapy). 
• 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. 
Stopping Therapy  
Your doctor may decide to stop your therapy for the following reasons: 
• if he/she believes that it is in your best interest 
• if your disease comes back during treatment 
• if you have side effects from the treatment that your doctor thinks are too severe 
• if new information shows that another treatment would be better for you 


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-0151 
CONTINUATION: page 8 of 12 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 
 
 
In this case, you will be informed of the reason therapy is being stopped. 
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. 
If you decide at any time to withdraw your consent to participate in the trial, we will not collect 
any additional medical information about you. If you withdraw your consent and leave the trial, 
any samples of yours that have been obtained for the study and stored at the NCI can be 
destroyed upon request. However, any samples and data generated from the samples that have 
already been distributed to other researchers or placed in the research databases cannot be 
recalled and destroyed. 
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 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. 
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. 
Certificate of Confidentiality  
To help us protect your privacy, we have obtained a Certificate of Confidentiality. The 
researchers can use this Certificate to legally refuse to disclose information that may identify you 
in any federal, state, or local civil, criminal, administrative, legislative, or other proceedings, for 
example, if there is a court subpoena. The researchers will use the Certificate to resist any 
demands for information that would identify you, except as explained below. 
You should also know that there are several circumstances in which the Certificate does not 
provide coverage. These include when information:  


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-0151 
CONTINUATION: page 9 of 12 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 be used for auditing or program evaluation internally by the NIH; or  
• must be disclosed to meet the legal requirements of the federal Food and Drug 
Administration (FDA).  
• is necessary for your medical treatment and you have consented to this disclosure;  
• is for other research.  
In addition, identifiable, sensitive information protected by this Certificate cannot be admissible 
as evidence or used for any purpose in any action, suit, or proceeding without your consent.  
You should understand that a Certificate of Confidentiality does not prevent you or a member of 
your family from voluntarily releasing information about yourself or your involvement in this 
research. If an insurer, employer, or other person obtains your written consent to receive research 
information, then the researchers will not use the Certificate to withhold that information. 
The Certificate of Confidentiality will not protect against the required reporting by hospital staff 
of information on suspected child abuse, reportable communicable diseases, and/or possible 
threat of harm to self or others. 
Conflict of Interest 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of interest. This process is detailed in a Protocol Review Guide. 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.  
Use of Specimens and Data for Future Research  
To advance science, it is helpful for researchers to share information they get from studying 
human samples. They do this by putting it into one or more scientific databases, where it is stored 
along with information from other studies. A researcher who wants to study the information must 
apply to the database and be approved. Researchers use specimens and data stored in scientific 
databases to advance science and learn about health and disease. 
We plan to keep some of the specimens and data that we collect and use them for future research 
and share them with other researchers. We will not contact you to ask about each of these future 
uses. These specimens and data will be stripped of identifiers such as name, address or account 
number, so that they may be used for future research on any topic and shared broadly for research 
purposes.. Your specimens and data will be used for research purposes only and will not benefit 
you. It is also possible that the stored specimens and 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.  


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-0151 
CONTINUATION: page 10 of 12 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 
 
If you do not want your stored specimens and data used for future research, please contact us in 
writing and let us know that you do not want us to use your specimens and/or data. Then any 
specimens that have not already been used or shared will be destroyed and your data will not be 
used for future research. However, it may not be possible to withdraw or delete materials or data 
once they have been shared with other researchers.  
 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 12-C-0151 
CONTINUATION: page 11 of 12 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, David S. Schrump, M.D., Building 10, Room 4-3940, Telephone: 240-760-6239. 
You may also call the Clinical Center Patient Representative at 301-496-2626. If you have any 
questions about the use of your tissue for future research studies, you may also contact the Office 
of the Clinical Director, Telephone: 240-760-6070. 
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-0151 
CONTINUATION: page 12 of 12 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 AUGUST 06, 2018 THROUGH AUGUST 05, 2019.  
 
 
 
 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
 
Print Name 
 
 
Print Name