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Patient cohorts will receive 24h infusions of mithramycin targeting total doses previously administered during 7 daily six hour infusions at 30-50 mcg/kg.  
The 24h infusions will be administered every 14 days (1 cycle). Four cycles will constitute one course of therapy.  
Pharmacokinetics and toxicity assessment to define MTD will be assessed during Cycle 1 of the first course of therapy.  
Due to uncertainties regarding potential cumulative toxicities, no intra-patient dose escalation will be allowed.  
Once MTD has been defined, patients will be enrolled into two cohorts (primary thoracic malignancy vs neoplasm of non-thoracic origin metastatic to the chest) to determine clinical response rates at the MTD, using a Simon Optimal Two Stage Design for Phase I Clinical Trials targeting an objective response rate (RECIST) of 30%.  
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 4 of the first cycle of therapy for analysis of pharmacodynamic endpoints. Optional tumor biopsies may be requested at the completion of Course 1 (4 cycles) and in patients exhibiting objective responses.  
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| Criteria for Removal from Protocol Therapy ................................................................30                               |                                                                                                                                                         |
|---------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------|
| 3.6.1                                                                                                                                       |                                                                                                                                                         |
| 3.6.2                                                                                                                                       | OFF-Study Criteria ............................................................................................................................31       |
| CONCOMITANT MEDICATIONS/MEASURES .....................................................................31                                    |                                                                                                                                                         |
| Hematologic and Blood Product Support ..............................................................................31                      |                                                                                                                                                         |
| 4.1                                                                                                                                         |                                                                                                                                                         |
| 4.2                                                                                                                                         | Antiemetic Therapy and Transaminitis Prophylaxis ..................................................31                                                   |
| 4.3                                                                                                                                         | Antidiarrheals ..........................................................................................................................31             |
| 4.4                                                                                                                                         | Electrolyte Replacement .........................................................................................................31                     |
| CORRELATIVE STUDIES / BIOSPECIMEN COLLECTION .............................................31                                                |                                                                                                                                                         |
| Pharmacokinetic (PK) Sampling ........................................................................................31                    |                                                                                                                                                         |
| 5.1                                                                                                                                         |                                                                                                                                                         |
| 5.2                                                                                                                                         | Studies on Tumor Tissue .......................................................................................................31                       |
| 5.3                                                                                                                                         | Sample Storage, Tracking and Disposition .....................................................................32                                        |
| Thoracic Epigenetics Laboratory ......................................................................................32                    |                                                                                                                                                         |
| 5.3.1                                                                                                                                       |                                                                                                                                                         |
| 5.3.2                                                                                                                                       | Blood Processing Core (BPC) ...........................................................................................32                               |
| Protocol Completion/Sample Destruction ........................................................................33                           |                                                                                                                                                         |
| 5.3.3                                                                                                                                       |                                                                                                                                                         |
| DATA COLLECTION AND EVALUATION                                                                                                              |                                                                                                                                                         |
| 6.1                                                                                                                                         | DATA COLLECTION .....................................................................................................................................34 |
| 6.1.1                                                                                                                                       | Routine Data Collection                                                                                                                                 |
| 6.1.2                                                                                                                                       | Exclusions to Routine Data Collection ............................................................................35                                    |
| 6.2                                                                                                                                         | DATA SHARING Plans                                                                                                                                      |
| 6.2.1                                                                                                                                       | Human Data Sharing Plan................................................................................................35                               |
| Genomic Data Sharing Plan                                                                                                                   |                                                                                                                                                         |
| 6.2.2                                                                                                                                       |                                                                                                                                                         |
| 6.3                                                                                                                                         | Response Criteria ............................................................................................................................36        |
| 6.3.1                                                                                                                                       | Disease Parameters ..............................................................................................................36                     |
| 6.3.2                                                                                                                                       | Methods of Evaluation of Measurable Disease .........................................................37                                                 |
| 6.3.3                                                                                                                                       | Response Criteria for Radiographic Studies ...............................................................38                                            |
| Confirmatory Measurement/Duration of Response .....................................................40                                       |                                                                                                                                                         |
| 6.3.4                                                                                                                                       |                                                                                                                                                         |
| 6.4                                                                                                                                         | Toxicity Criteria                                                                                                                                       |
| NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN….41                                                                              |                                                                                                                                                         |
| Definitions .............................................................................................................................41 |                                                                                                                                                         |
| 7.1                                                                                                                                         |                                                                                                                                                         |
| 7.2                                                                                                                                         | OHSRP Office of Compliance and Training/IRB Reporting ......................................41                                                          |
| 7.2.1                                                                                                                                       | Expedited Reporting ...............................................................................................................41                   |
| 7.2.2                                                                                                                                       | IRB Requirements for PI Reporting Continuing Review ..........................................41                                                        |
| 7.2.3                                                                                                                                       | NCI Clinical Director Reporting....................................................................................41                                   |
| 7.3                                                                                                                                         | NIH Required Data and Safety Monitoring Plan............................................................41                                              |
| 7.3.1                                                                                                                                       | Principal Investigator/Research Team .............................................................................41                                    |
| SPONSOR SAFETY REPORTING .....................................................................................................42            |                                                                                                                                                         |  
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14.1.1 Table 1: Genes Differentially Expressed In Vitro and In Vivo by Mithramycin ..58 14.1.2 Table 2: Tissue Mithramycin Concentrations in Mice Following IV or IP
Injection 14.2 Figures ....................................................................................................................................60 14.2.1 Figure 1: Effects of CSC on ABCG2 Expression in Lung and Esophageal CANCERS
14.2.2  Figure 2: Role of AhR, Sp1 and Nrf2 in ABCG2 Activation by CSC. .................62 14.2.3 Figure 3: Effects of Mithramycin in Lung and Esophageal Cancer and Malignant Pleural Mesothelioma (MPM) Cells 14.2.4 Figure 4: Microarray Analysis of Mithramycin Effects on Gene Expression Cultured A549 and Calu-6 Cells, and A549 Xenografts Related to Respective Controls 14.2.5 Figure 5: Ingenuity Pathway Analysis Depicting Representative Cancer Network Targeted by Mithramycin in Tumor Xenografts Following Systemic Administration of Mithramycin in Athymic Nude Mice. .......................................68 14.2.6  Figure 6: Effects of MM on Cell Cycle Progression, Senescence and Apoptosis in MPM Cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001). ......................................70 14.2.7  Figure 7: AST and ALT Levels and Representative Liver Biopsy from Patients with Mithramycin Mediated Hepatotoxicity. Transaminitis was Associated with Apoptotic Hepatocyte Death......................................................................................72 14.2.8 Figure 8: Results of DMET Analysis Demonstrating that Mithramycin Mediated Transaminitis Correlates with SNPs in ABCB4 and ABCB11. .............74 14.2.9  Figure 9: Results of DMET Analysis Depicting Associations Between AST and ALT Elevations with SNPs in ABCB4, ABCB11, PEPT2, and RALB from Patients Receiving Mithramycin 14.2.10 Figure 10: Simulated Mithramycin Plasma Levels During and After 24 Hour Infusion Based on Direct Quantitation of Mithramycin Plasma Levels from the Recent Phase II Mithramycin Trial (Grey Dots).............................................................77 14.2.11 Figure 11: Observed and Predicted Mithramycin Plasma Levels Following 24 Hour Mithramycin Infusions 14.3 Appendices ..................................................................................................................................79 14.3.1 Appendix 1: Mithramycin Clinical Experience Reported in the Literature...........79 14.3.2  Appendix 2: Study Calendar............................................................................................84 14.3.3 Appendix 3: Sample Pharmacokinetic and EKG Worksheet for Mithramycin.....88 14.3.4  Appendix 4: Sample Patient Log for Reactions/Symptoms ....................................92 14.3.5  Appendix 5: Sample Patient Log for Concomitant Medication...............................93 14.3.6  Appendix 6: Sample Monitoring Toxicities Between Mithramycin Cycles with Local Physician
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:8' genes mediating oncogene signaling, cell cycle regulation, senescence and apoptosis (Rao et al, submitted for publication).  
Our preclinical studies have clearly shown that 24 hour mithramycin treatment is sufficient to mediate progressive decreases in cancer cell viability in vitro ; furthermore, repeated IP administration of mithramycin (qM-W-F x 3 weeks) significantly inhibits growth of subcutaneous lung and pleural mesothelioma xenografts. Our micro-array experiments analyses (performed immediately following 24h mithramycin in vitro , or approximately 3 days following the last IP treatment of mice bearing tumor xenografts) demonstrated considerable overlap between in vitro and in vivo mithramycin exposures. These findings strongly suggest that the anti-proliferative effects of mithramycin are not primarily due to acute cytotoxicity, but are instead related to transcriptional reprogramming, which persists long after mithramycin exposure. These observations 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 our 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. For example, Sp1 is over-expressed in gastric, colon, breast, pancreas and prostate cancers ( 5458 ) 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 , 5863 ); 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 Spl 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.
untitled h1
preclinical toxicology 1.3.2

In mice, the LD 50 was determined to be about 2000 mcg/kg (6000 mcg/m 2 ) 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/m 2 ) and monkeys (1200 mcg/m 2 ) ( 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 (20) as well as those reported by Grohar et al ( 65 ), indicate that IP doses of 1-2 mg/kg/day 3 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.  
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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 god activity in 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 ).
untitled h1
1.3.4 recent experience with mithramycin

In the recent Phase II trial (12C0151) in TSB/CCR/NCI, 12 patients (10 males; 2 females; median age 51y; range 40-72y) with refractory thoracic malignancies (6 MPMs, 2 esophageal cancers, 2 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 were 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 25 mcg/kg/infusions. This toxicity always occurred on Day 4 of the infusion. Percutaneous biopsies from three individuals revealed apoptotic hepatocellular death (Figure 7A). Four patients (2 with M) had no hepatotoxicity and tolerated dose escalation to 30 mcg/kg/infusion. Peak and steady state mithramycin levels were 20 and 8 nM, respectively, which were not different in patients with or without hepatotoxicity (Figure 7B).  
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transporters are involved in hepatocellular bile accumulation. Accordingly, the enrollment criteria of the clinical trial is now amended to include the following genotypes: RALBP rs12680 (GG),
CYP8B1 rs6774801 (CC or CA), ABCB4 rs2302387 (CC), and ABCB11 rs4668115 (GG) (Figure 9I-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. Plasma mithramycin levels measured in the 12 patients enabled simulation of pharmacokinetics of mithramycin administered via 24h infusions (Figure 10). Based on these data, doses in the range of 25-200 mcg/kg are estimated to produce plasma levels mediating pronounced antitumor effects in preclinical murine xenograft experiments.
untitled h1
1.3.4.2 current trial

In the current trial (16C0152), 3 patients with stage IV malignancies received 24-hour mithramycin infusions at 60 mcg/kg.  Patient \#1 with HLRCC exhibited Grade 4 AST/ALT elevations immediately following completion of his cycle infusion.  AST/ALT levels peaked at over 6000, and were associated with Grade 2 nausea and vomiting.  This transaminitis resolved without treatment over the next 7-10 days. Patients \#2 and 3 both of whom had stage IV esophageal adenocarcinoma tolerated their first infusions well with no increase in AST/ALT until approximately 48 hours post-infusion when they developed Grade 4 transaminitis (peak ~2500)
which was also associated with Grade 1-2 nausea and vomiting.  The transaminitis in both patients resolved in approximately 7 days whereas the nausea and vomiting resolved within approximately 24 hours.  
Due to the severity of the dose limiting transaminitis, Patient \#1 requested to be removed from study to pursue other investigational therapy.  He was removed from protocol treatment and study when appropriate criteria were met.  Patients \#2 and 3 were dose reduced for subsequent cycles, receiving mithramycin at 30 mcg/kg per infusion with no significant AST/ALT elevations. Interestingly, both of these patients had stable disease by imaging criteria at initial treatment and evaluation following the completion of the first course of therapy.  Both of these patients appeared to exhibit clinical benefit from their treatment, as evidenced by significant resolution of dysphagia/odynophagia, improved oral intake and overall sense of well-being, and have continued on study. PK results pertaining to Cycles 1 and 2 infusions in these patients are summarized in Figure 11, and closely approximate predicted levels based on modeling from drug levels obtained in the Phase II mithramycin study (12C0151). The lack of significant liver toxicities observed in Patients \#2 and 3 who received mithramycin infusions at 30 mcg/kg per infusion and possible clinical benefit warrant evaluation of additional patients at this dose level, and escalation if appropriate to a new intermediate level (45 mcg/kg per infusion), to optimize mithramycin delivery to solid tumors.
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3 study implementation 3.1 study

This Phase I/II study is designed primarily to evaluate the toxicities, pharmacokinetics and clinical efficacy of mithramycin administered as a continuous 24-hour infusion in patients with thoracic malignancies. All patients will be evaluated initially in the Thoracic Surgery Clinic, Clinical Center, NIH. Once appropriate consents have been signed, patients will undergo biopsy of their tumors for analysis of pharmacodynamics endpoints via endoscopic procedures performed in the operating room by the Thoracic Surgery attending physicians or by percutaneous FNA methods performed by interventional radiology staff at the Clinical Center, NIH. Specimens will be processed for DNA/RNA isolation, and immunohistochemistry studies. If sufficient materials are available from a previous biopsy either at the NIH or elsewhere, this baseline biopsy may be omitted as outlined in Appendix 2. Patients will be hospitalized on 3NW, NIH Clinical Center, and receive 24h infusions of mithramycin as outlined below using a conventional Phase I study design, with no intra-patient dose escalation, and no routine cytokine support. Treatment cycles will be separated by 2 weeks to allow recovery from systemic toxicities. Due to potential cardiac toxicity related to mithramycin, all patients will have EKGs (12 lead) prior to, and daily EKGs for 4 days following mithramycin infusions (first cycle only unless otherwise clinically indicated), and will be on telemetry during the Cycle 1 infusion (and subsequent cycles if clinically indicated per Cardiology recommendations). Patients will also have routine bloodwork as outlined in Section 3.5. Blood will be obtained at designated time points to assess mithramycin levels are outlined in Section 5.1. Unless medical difficulties arose during the collection of the first biopsy, a posttreatment tumor biopsy will be obtained on Cycle 1 Day 4 (+/- 3 days) to assess molecular endpoints. At the time of each biopsy session, representative samples will be evaluated by frozen section techniques to verify the histology and quality of the tumor tissue for subsequent analysis.  
An attempt will be made to biopsy the same tumor site for consistency of sampling during the treatment cycles. Approximately 14 days following initiation of the first treatment cycle, a second treatment cycle will commence. If possible, an optional tumor biopsy may be taken at treatment evaluation following the end of Course 1, and additional optional tumor biopsies requested if a patient experiences a dramatic clinical response to treatment, provided that the biopsies are easy to obtain and pose little risk to the patients (refer to Section 5.2 and Appendix 2). CT guided research biopsies will not exceed 5 per year on this study.  At the end of the fourth cycle (approximately 56-60 days following the initiation of the first treatment cycle), patients will return to the Thoracic Surgery Clinic for repeat staging studies and blood work to analyze treatment response using criteria outlined in Section 6.3.3. In patients who exhibit response to therapy and commence a second course of mithramycin treatment an additional tissue biopsy may be requested during this course of therapy for confirmatory analysis of pharmacodynamics endpoints identified in preclinical experiments to be associated with regressions of human cancer xenografts in mice.  
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following Course 1. If insufficient tissue was obtained or if there was harm associated with 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 (refer to Appendix 2). 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 focused microarray and/or qRT-PCR analysis of ~50 genes modulated by mithramycin in murine xenograft experiments. 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 under direction of the PI.  
Note: If there is an optional biopsy for research in the protocol, then the patient will consent 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.  
* May be omitted as outlined in Appendix 2: Study Calendar.
untitled h1
5.3 sample storage tracking and

Samples will be ordered in CRIS and tracked through Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed.  Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required.
untitled h1
5.3.1 thoracic epigenetics laboratory

This study will be conducted within the Thoracic Surgery Branch, 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 in the Figg Lab, GMB.  
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 the Thoracic Surgery Branch, NCI with access limited to the PI or study coordinator.  
Focused gene expression microRNA, and DNA methylation signatures will be analyzed in Dr.  
David Schrump's laboratory using a panel of genes and miRs that were noted to be modulated in subQ cancer xenografts following IP mithramycin treatment.
untitled h1
5.3.2 blood processing core (bpc)

Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred).  
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+2 If the patient withdraws consent the participants data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved.  
Following completion of this study, if the subject has co-enrolled on 06C0014, samples will be transferred to the Tissue Procurement protocol and remain in storage in the Blood Processing Core
(BPC) as detailed within 06C0014. Access to these samples will only be granted following IRB
approval of an additional protocol, granting the rights to use the material.  If the subject has not co-enrolled on the Tissue Procurement protocol, the samples will be destroyed.  
The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of Section 7.2.
untitled h1
data collection and evaluation 6

For the purposes of the research sample analyses and correlation with clinical outcomes, demographic information, histology, operative and peri-operative interventions, pathologic findings, laboratory and imaging parameters (performed as part of routine or protocol specified patient care) may be collected on this study. The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D, Labmatrix) and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts.  All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant.  
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event.  
Document AEs from the first study intervention, Study Day 1, through 2-4 weeks after the last dose of study therapy. Beyond 2-4 weeks after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded. An abnormal laboratory value will be considered an AE only if the laboratory abnormality is characterized by any of the following:
.  
Results in discontinuation from the study  
.  
Is associated with clinical signs or symptoms
.  
Requires treatment or any other therapeutic intervention
.  
Is associated with death or another serious adverse event, including hospitalization
.  
Is judged by the Investigator to be of significant clinical impact
.  
If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
End of Study Procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
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6.3.2.2 metastatic bone lesions

Disease progression is considered if any new lesion 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 ( 80 ).
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6.3.2.3 clinical lesions

Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and ≥ 10 mm diameter as assessed using calipers (e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.
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6.3.2.4 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.
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6.3.2.5

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.
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6.3.3 response criteria for radiographic

6.3.3.1   Measuring of Soft Tissue Disease a. Evaluation of Target Lesions
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complete response (cr)

Disappearance of all target lesions. Any pathological lymph nodes (whether target or nontarget) must have reduction in short axis to < 10 mm.
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partial response (pr)

At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.
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progressive disease (pd)

At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progressions.)
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Informed consent process
.  
Eligibility confirmation
.  
Drug administration and accountability
.  
Adverse events monitoring Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating cnter.  
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.
untitled h1
10 statistical considerations

The Phase 1 dose escalation study of mithramycin infusion will be conducted as a single arm study to minimize the number of patients needed to define MTD. Pharmacokinetic analyses, as well as analyses of molecular endpoints such as HDAC4, KIAA1199, PMAIP and p21 expression in tumor tissues and PBMC before and after treatment will be performed, with the primary focus being with respect to results at the MTD. 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 trial and will be based on limited nmbers of patients, the analyses will be considered exploratory and hypothesis generating rather than definitive. As many as 15 patients may theoretically need to be accrued to define the MTD if the amended dose levels (DL-1 and DL-1A) each require 6 patients, in addition to the 3 subjects that were already enrolled at dose level DL1. The primary endpoint of the Phase II component study will be evaluated for objective response by RECIST criteria v 1.1. Patients will be enrolled into 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 cohort and if 0 of the 7 have a response, then no further patients will be accrued in that cohort. If 1 or more of the first 7 in a cohort have a response, then accrual will 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%.  
The following table summarizes the two stage design that will be used for each cohort in the Phase I component of this trial.  
| Cumulative                          |                      |                                        |
|-------------------------------------|----------------------|----------------------------------------|
| Number of                           | Decision             |                                        |
| Responses                           |                      |                                        |
| Stage 1: Enter 7 patients           | 0                    | Terminate the trial: agent ineffective |
| Inconclusive result, continue trial |                      |                                        |
| l or more                           | (proceed to Stage 2) |                                        |
| Stage 2: Enter 14 additional        | 2 or less            | Terminate the trial: agent ineffective |
| patients                            | 3 or more            | Terminate the trial: agent effective   |  
With two cohorts, up to 42 evaluable patients may be required to be accrued to the Phase II portion.  
It is expected that 10-15 patients per year may enroll onto this study. Thus, 2 to 3 years may be neded to enroll up to 15 patients in Phase I plus 36 evaluable patients in Phase II (up to 6 patients may continue from the Phase I component of the trial). This may be completed much sooner depending on the number actually required. To allow for a small number of inevaluable patients, the accrual ceiling will be set at 60.
untitled h1
10.1 m ethods of a

Response rates will be calculated as the percent of patients whose best response is a CR or PR.  
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.
untitled h1
10.2 e valuation of r

Any patient who is enrolled and receives at least one full course (4 cycles) 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 completing a full course of mithramycin and do not expire within 14 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 will be taken off-treatment and will only be evaluable for toxicity (not evaluable for response).
untitled h1
10.3 secondary endpoints

Pharmacokinetic analysis will be conducted using non-compartmental methods.  
For pharmacodynamic endpoints, descriptive statistics will be used for each endpoint. Briefly, 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 nonparametric 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.
untitled h1
1 human subjects protections 11.1

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.
untitled h1
1.2 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.
untitled h1
11.3 participation of subjects unable

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 11.4), all subjects ≥ age 18 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 a surrogate.  
For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in Policy 403 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.
untitled h1
11.4 e valuation of p

The risks and benefits of participation for adults who become unable to consent on study are no different than those described for the rest of the study population.  
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| p X^ I kkk qqqq                                                                                 |
| HIJIKKL M NMO                                                                                 |
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