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11.2 appendix 2 performance status

| PERFORMANCE STATUS CRITERIA Karnofsky and Lansky performance scores are intended to be multiples of 10.   |                                                |                                       |                                    |                                       |                                      |
|-----------------------------------------------------------------------------------------------------------|------------------------------------------------|---------------------------------------|------------------------------------|---------------------------------------|--------------------------------------|
| ECOG (Zubrod)                                                                                             | Karnofsky                                      | Lansky                                |                                    |                                       |                                      |
| Score                                                                                                     | Description                                    | Score                                 | Description                        | Score                                 | Description                          |
| 0                                                                                                         | Fully active, able to carry on all pre-disease | 100%                                  | Normal, no complaints, no          | 100%                                  | Fully active, normal.                |
| performance without restriction.                                                                          | evidence of disease.                           |                                       |                                    |                                       |                                      |
| 90%                                                                                                       | Able to carry on normal activity;              | 90%                                   | Minor restrictions in physically   |                                       |                                      |
| minor sighs of symptoms of                                                                                | strenuous activity.                            |                                       |                                    |                                       |                                      |
| disease.                                                                                                  |                                                |                                       |                                    |                                       |                                      |
| Restricted in physically strenuous activity but                                                           | 80%                                            | 80%                                   |                                    |                                       |                                      |
| l                                                                                                         | Able to carry on normal activity               | Active, but tires more quickly.       |                                    |                                       |                                      |
| ambulatory, able to carry out light or                                                                    | with effort; some signs or                     |                                       |                                    |                                       |                                      |
| sedentary work, e.g., light housework, office                                                             | symptoms of disease.                           |                                       |                                    |                                       |                                      |
| work.                                                                                                     | 70%                                            | Cares for self, unable to carry on    | 70%                                | Both greater restriction of, and less |                                      |
| normal activity or do active                                                                              | time spent in, play activities.                |                                       |                                    |                                       |                                      |
| work.                                                                                                     |                                                |                                       |                                    |                                       |                                      |
| 2                                                                                                         | Ambulatory and capable of all self-care but    | 60%                                   | Requires occasional assistance     | 60%                                   | Up and around, but minimal active    |
| unable to carry out any work activities. Up and                                                           | but is able to care for most of                | play; keeps busy with quieter         |                                    |                                       |                                      |
| about more than 50% of waking hours.                                                                      | own needs.                                     | activities.                           |                                    |                                       |                                      |
| 50%                                                                                                       | Requires considerable assistance               | 50%                                   | Gets dressed, but lies around much |                                       |                                      |
| and frequent medical care.                                                                                | of the day; no active play; able to            |                                       |                                    |                                       |                                      |
| participate in quiet play and                                                                             |                                                |                                       |                                    |                                       |                                      |
| activities.                                                                                               |                                                |                                       |                                    |                                       |                                      |
| 3                                                                                                         | Capable of only limited self-care, confined to | 40%                                   | Disabled; requires special care    | 40%                                   | Mostly in bed; participates in quiet |
| bed or chair more than 50% of waking hours                                                                | and assistance                                 | activities.                           |                                    |                                       |                                      |
| 30%                                                                                                       | Severely disabled;                             | 30%                                   | In bed; needs assistance even for  |                                       |                                      |
| hospitalization indicated.                                                                                | quiet play.                                    |                                       |                                    |                                       |                                      |
| although death not imminent.                                                                              |                                                |                                       |                                    |                                       |                                      |
| Completely disabled.  Cannot carry on any                                                                 | 20%                                            | 20%                                   |                                    |                                       |                                      |
| 4                                                                                                         | Very ill; hospitalization                      | Often sleeping; play entirely limited |                                    |                                       |                                      |
| self-care. Totally confined to a bed or chair                                                             | necessary;  active supportive                  | to very passive activities.           |                                    |                                       |                                      |
| treatment required.                                                                                       |                                                |                                       |                                    |                                       |                                      |
| 10%                                                                                                       | 10%                                            |                                       |                                    |                                       |                                      |
| Moribund, fatal process                                                                                   | No play; does not get out of bed               |                                       |                                    |                                       |                                      |
| progressing rapidly                                                                                       |                                                |                                       |                                    |                                       |                                      |
| s                                                                                                         | Dead                                           | 0%                                    | Patient expired                    | 0%                                    | Unresponsive; Dead                   |  
| 11.3 APPENDIX 3: STUDY CALENDAR   | Cycle 1 (28 Days) and subsequent cycles                                                                                                                      | Post Therapy  Follow-up   |     |     |     |     |     |     |     |     |     |     |    |    |
|-----------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|----|----|
| Procedurep                        | At least 30 days  after last  mithramycin dosem                                                                                                              |                           |     |     |     |     |     |     |     |     |     |     |    |    |
| Screening/ Baseline** Day         | Day                                                                                                                                                          | Day                       | Day | Day | Day | Day | Day | Day | Day | Day | Day | Day |    |    |
| 1                                 | 2                                                                                                                                                            | 3                         | 4   | 5   | 6   | 7   | 8   | 9   | 10  | 14  | 21  | 28  |    |    |
| History, PEa                      | X                                                                                                                                                            | Xa                        | Xa  | Xa  | Xa  | Xa  | Xa  |     |     |     |     |     |    |    |
| Weightaa                          | X                                                                                                                                                            | X                         | X   | X   | X   | X   | X   |     |     |     |     |     |    |    |
| Height (baseline only)            | X                                                                                                                                                            |                           |     |     |     |     |     |     |     |     |     |     |    |    |
| Vital signs (BP, HR, RR, temp)    | X                                                                                                                                                            | X                         | X   | X   | X   | X   | X   | X   |     |     |     |     |    |    |
| Performance Score                 | X                                                                                                                                                            | X                         | X   |     |     |     |     |     |     |     |     |     |    |    |
| Histologic Path confirmation      | X                                                                                                                                                            |                           |     |     |     |     |     |     |     |     |     |     |    |    |
| -                                 | CBC, differential                                                                                                                                            |                           |     |     |     |     |     |     |     |     |     |     |    |    |
| -                                 | sodium, potassium, chloride,  CO2, creatinine, glucose,  BUN, albumin, calcium,  magnesium, phosphorus, uric  acid LDH and (amylase,  lipase, baseline only) |                           |     |     |     |     |     |     |     |     |     |     |    |    |
| -                                 | alk phos, ALT/AST, T  bilirubin, Direct bilirubin, T  protein                                                                                                | X                         | Xb  | Xb  | Xb  | Xb  | Xb  | Xb  | Xb  | Xb  | Xb  | Xb  | Xb | X  |
| -                                 | Plasma sample (1mL)c                                                                                                                                         | Xc                        |     |     |     |     |     |     |     |     |     |     |    |    |
| -                                 | PT/PTT, Thrombin Time,  Fibrinogend                                                                                                                          | Xd                        | Xd  | Xd  | Xd  | Xd  | Xd  | Xd  | Xd  | X   |     |     |    |    |
| -                                 | HIV, HBV, HCVe                                                                                                                                               | Xe                        |     |     |     |     |     |     |     |     |     |     |    |    |
| -                                 | Pregnancy Test (urine)f                                                                                                                                      | Xf                        | Xf  |     |     |     |     |     |     |     |     |     |    |    |  
| Cycle 1 (28 Days) and subsequent cycles                                                              | Post Therapy  Follow-up At least 30  days after last  mithramycin  dosem   |                   |        |        |           |        |         |         |         |
|------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------|-------------------|--------|--------|-----------|--------|---------|---------|---------|
| Procedurep                                                                                           | Screening/                                                                 | Baseline** Day  1 | Day  2 | Day    |           |        |         |         |         |
| y 3                                                                                                  | 4                                                                          |                   |        |        |           |        |         |         |         |
| Da                                                                                                   | Day  5                                                                     | Day  6            | Day  7 | Day  8 | Day  9    | Day 10 | Day  14 | Day  21 | Day  28 |
| -                                                                                                    | Urinalysis                                                                 | Xff               | Xff    |        |           |        |         |         |         |
| -                                                                                                    | Coags: , PFA-100, VWF                                                      |                   |        |        |           |        |         |         |         |
| antigen, VWF activity, Factor  VIII activity, D-dimer, fibrin  degredation products, FXIII  activity | X                                                                          |                   |        |        |           |        |         |         |         |
| Mithramycin infusion                                                                                 | X                                                                          | X                 | X      | X      | X         | X      | X       |         |         |
| Receive oral/IV dexamethasoneo                                                                       | X                                                                          | X                 | X      | X      | X         | X      | X       | X       |         |
| MUGA or echocardiogram                                                                               | X                                                                          |                   |        |        |           |        |         |         |         |
| 12-lead EKGn                                                                                         | X                                                                          | X                 | Xn     | X      | X         |        |         |         |         |
| Radiological Assessments: CT,  MRI, FDG-PET, Bone scang                                              | X                                                                          | Xg                | X      |        |           |        |         |         |         |
| Xh                                                                                                   |                                                                            |                   |        |        |           |        |         |         |         |
| Pharmacokineticsh                                                                                    | Xh                                                                         | Xh                | Xh     | Xh     | (and  D9) |        |         |         |         |
| Serum Pharmacodynamic Markeri                                                                        | Xi                                                                         |                   |        |        |           |        |         |         |         |
| Mandatory biopsy for NR0B1  expressionj                                                              | Xj                                                                         | Xj                |        |        |           |        |         |         |         |
| Adverse Eventsk                                                                                      | X                                                                          | X                 |        |        |           |        |         |         |         |
| Concomitant Medicationsl                                                                             | X                                                                          | X                 |        |        |           |        |         |         |         |  
**Blood tests within 7 days prior to enrollment; imaging studies within 2 weeks prior to enrollment (UNLESS otherwise stated), histological path evaluation is recommended to be completed prior to arrival aWeekly during active treatment, and weekly until recovered from toxicity.  
aaDaily weight recommended for days 2-7 of mithramycin infusion.  
bDaily on days 2 through7, then twice weekly days 9 to 28 of cycles 1 through 3 c1 mL of plasma will be obtained, filtered through a 0.22 micron filter, frozen and saved by hematology pathology for future reference if needed, prior to receiving drug on day 1 of cycle 1 and 2 only. See **Error! Reference source not found.** for details.  
dCoagulation studies: drawn on days 1, (3, 4 or 5) and (7, 8 or 9) or as clinically indicated . If screening coagulation studies drawn within 1 week of starting drug, day 1 can be omitted for cycle 1 only.  
eExcluded by history only. No laboratory testing of HIV, HBV or HCV is required.  
frequired at screening/prior to cycle 1 and prior to each subsequent cycle on females of childbearing potential ffrequired at screening/prior to cycle 1 and prior to each subsequent cycle gperformed after every EVEN numbered treatment cycle, i.e. cycle 2, cycle 4, etc until off therapy and then every two months until off study or as indicated by clinical response criteria see (section 6.2). Imaging studies will be sent to NCI POB for volumetric analysis (see **Error! Reference source not found.** for details). hPK samples: prior to and at the completion of the first infusion, then trough and end of infusion samples with day 4 and 7 doses, and 24 and 48 hours after day 7 dose. Samples will therefore be drawn on days 1, 2, 4, 7, 8, 9 as indicated in **Error! Reference source not found.** and **Error! Reference source not**
found.. iA serum sample for potential biomarker analysis will be obtained prior to the first mithramycin injection, and then at the time of each restaging studies. See Error! Reference source not found. for details.  
jEligibility and timing for mandatory biopsy is defined in **Error! Reference source not found.**. kSee **Error! Reference source not found.** to obtain sheet to record adverse events at home lSee **Error! Reference source not found.** to record all medications taken at home mPost therapy evaluations will be completed until at least 30 days after the last dose of mithramycin and until resolution of toxicity. nAll EKGs should be done prior to infusion. EKG on day 4 to be done on cycle 1 only.  
o2.5 mg dexamethasone/m2 BSA PO or IV starting 12 hours before the first dose of mithramycin, then prior to the first dose, and then twice daily for 24 hours after the last dose of mithramycin pFor patients who have discontinued mithramycin infusions during any cycle prior to completion of the 7 daily infusions, the required laboratory studies and other evaluations can be adjusted by the principal investigator or associate investigator as necessary.  
11.4 APPENDIX 4: PHARMACOKINETIC WORKSHEET FOR MITHRAMYCIN (PHASE I **AND PHASE**
II) **FOR NURSING STAFF**  
| Patient ID         | Weight (kg), height (cm), BSA (m2 )            |             |             |           |
|--------------------|------------------------------------------------|-------------|-------------|-----------|
| Dose level (mg/kg) | Total dose (mg)                                |             |             |           |
| Date:              | Infusion start time Infusion stop time         |             |             |           |
| Pre-dose EKG time: | Signature of person completing  EKG:           |             |             |           |
| Sample #           | Hour                                           | Target Time | Actual Time | Signature |
| 1                  | pre dose                                       |             |             |           |
| 2                  | 3 h post-start of  infusion                    |             |             |           |
| 3                  | Just prior to the  end of the 6 hour  infusion |             |             |           |
| 4                  | 0.25 h postinfusion                                                |             |             |           |
| 5                  | 0.5                                            |             |             |           |
| 6                  | 1                                              |             |             |           |
| 7                  | 2                                              |             |             |           |
| 8                  | 3                                              |             |             |           |
| 9                  | 5                                              |             |             |           |
| 10                 | 7                                              |             |             |           |
| 11*                | 9-12                                           |             |             |           |
| *If feasible       |                                                |             |             |           |
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11.2 appendix 2 performance status

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

| Sample #   | Hours                      | Target Time   | Actual Time   | Signature   |
|------------|----------------------------|---------------|---------------|-------------|
| 1          | 24 hours after  day 7 dose |               |               |             |  
2 24.5 hours after  
| day 7 dose   |
|--------------|  
Day 9* Date:  
| Sample #      | Hours                        | Target Time   | Actual Time   | Signature   |
|---------------|------------------------------|---------------|---------------|-------------|
| * 1           | 48 hours after  day 7 dose   |               |               |             |
| * 2           | 48.5 hours after  day 7 dose |               |               |             |
| * If feasible |                              |               |               |             |  
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line). Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant. Tubes should then be placed on crushed ice, and samples should be centrifuged for 15 minutes at approximately 1000 x g at 0-5 °C within 2 hr after collection. The plasma should be transferred to separate pre-labeled screw-capped polypropylene transfer tubes and stored at -80°C. Samples will be transported on dry ice to:
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diane cole

Pharmacology and Experimental Therapeutics Section Pediatric Oncology Branch, NCI
10 Center Drive, Building 10, room 1-5750.  
Bethesda, MD 201892-1101.  
Phone: 301-496-1757 E-mail: coled@mail.nih.gov Please notify Ms. Cole by e-mail or phone prior to transport of the samples.
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11.5 appendix 5 pharmacokinetic worksheet

| DAY 1 Patient ID   | Weight (kg), height (cm), BSA (m2 )             |               |                    |         |           |
|--------------------|-------------------------------------------------|---------------|--------------------|---------|-----------|
| Dose level (mg/kg) | Total dose (mg)                                 |               |                    |         |           |
| Date:              | Infusion start time Infusion stop time          |               |                    |         |           |
| Sample             | Hour                                            | Time Specimen | Time in Centrifuge | Time in | Signature |
| #                  | in Lab                                          | Freezer       |                    |         |           |
| 1                  | pre dose                                        |               |                    |         |           |
| 2                  | 3 h post-start  of infusion                     |               |                    |         |           |
| 3                  | Just prior to  the end of the  6 hour  infusion |               |                    |         |           |
| 4                  | 0.25 h postinfusion                                                 |               |                    |         |           |
| 5                  | 0.5                                             |               |                    |         |           |
| 6                  | 1                                               |               |                    |         |           |
| 7                  | 2                                               |               |                    |         |           |
| 8                  | 3                                               |               |                    |         |           |
| 9                  | 5                                               |               |                    |         |           |
| 10                 | 7                                               |               |                    |         |           |
| 11*                | 9-12                                            |               |                    |         |           |
| *If feasible       |                                                 |               |                    |         |           |  
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line). Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant. Tubes should then be placed on crushed ice, and samples should be centrifuged for 15 minutes at approximately 1000 x g at  
| polypropylene transfer tubes and stored at -80°C until shipped to the analytical site. Patient ID Weight (kg), height (cm), BSA (m2 ) Dose level (mg/kg) Total dose (mg)   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Day 2 Date: Infusion start time: Stop time:  
| Sample #   | Hours            | Time Specimen in   | Time in   | Time in   | Signature   |
|------------|------------------|--------------------|-----------|-----------|-------------|
| Lab        | Centrifuge       | Freezer            |           |           |             |
| 1          | pre dose         |                    |           |           |             |
| 2          | End of  infusion |                    |           |           |             |  
Day 4 Date: Infusion start time: Stop time:  
| Sample   | Hours            | Time Specimen   | Time in   | Time in   | Signature   |
|----------|------------------|-----------------|-----------|-----------|-------------|
| #        | in Lab           | Centrifuge      | Freezer   |           |             |
| 1        | pre dose         |                 |           |           |             |
| 2        | End of  infusion |                 |           |           |             |  
Day 7 Date: Infusion start time: Stop time:  
| Sample   | Hours            | Time Specimen   | Time in   | Time in   | Signature   |
|----------|------------------|-----------------|-----------|-----------|-------------|
| #        | in Lab           | Centrifuge      | Freezer   |           |             |
| 1        | pre dose         |                 |           |           |             |
| 2        | End of  infusion |                 |           |           |             |
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day 8 date

| Sample     | Hours                      | Time Specimen   | Time in   | Time in   | Signature   |
|------------|----------------------------|-----------------|-----------|-----------|-------------|
| Centrifuge | Freezer                    |                 |           |           |             |
| #          | in Lab                     |                 |           |           |             |
| 1          | 24 hours after  day 7 dose |                 |           |           |             |  
| 2   | 24.5 hours  after day 7  dose   |
|-----|---------------------------------|  
Day 9* Date:  
| Day 9* Date: Sample #   | Hours                         | Time Specimen   | Time in   | Time in   | Signature   |
|-------------------------|-------------------------------|-----------------|-----------|-----------|-------------|
| in Lab                  | Centrifuge                    | Freezer         |           |           |             |
| * 1                     | 48 hours  after day 7  dose   |                 |           |           |             |
| * 2                     | 48.5 hours  after day 7  dose |                 |           |           |             |
| * If feasible           |                               |                 |           |           |             |  
Collect 3 mL blood in a lavender top tube. Blood has to be drawn from a site away from the site of infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line).  
Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant. Tubes should then be placed on crushed ice, and samples should be centrifuged for 15 minutes at approximately 1000 x g at 0-5 °C within 2 hr after collection. The plasma should be transferred to separate pre-labeled screw-capped polypropylene transfer tubes and stored at -80°C. Samples will be transported on dry ice to: Diane Cole Pharmacology and Experimental Therapeutics Section Pediatric Oncology Branch, NCI 10 Center Drive, Building 10, room 1-5750. Bethesda, MD 201892-1101.  
Phone: 301-496-1757 E-mail: coled@mail.nih.gov Please notify Ms. Cole by e-mail or phone prior to transport of the samples.
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11.6 appendix 6 serum sample

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

a) Archival paraffin embedded tumor tissue for confirmation of EWS/FLI1 fusion transcript to determine eligibility for phase II portion of trial, and for evaluation of NR0B1 expression in patients with Ewing sarcoma Archival paraffin embedded tissue, or if paraffin embedded tissue is not available, 5 consecutive unstained slides, should be sent via express carrier to:
Brigitte Widemann, MD NCI, POB 10 Center Drive, Building 10 CRC Room 1-5750, MSC 1101 Bethesda, MD 20892 Phone: 301-496-7387 Please notify Dr. Widemann by e-mail (widemanb@mail.nih.gov) prior to shipment. Dr. Widemann will submit these samples to the Laboratory of Pathology for testing.
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tumor tissue from mandatory tumor

In patients with Ewing sarcoma and EWS-FLI1 fusion transcript ≥ 18 years old, who have disease amenable to percutaneous biopsy, and who meet other eligibility criteria for biopsy, a biopsy will be obtained prior to treatment and at steady state on treatment during cycle 1. Patients who meet these criteria will have to agree to the biopsies in order to being able to participate in the trial.
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tumor biopsies

Willingness to undergo tumor biopsies is required for patients with disease amenable to percutaneous biopsy on this protocol because modulation of molecular targets in tumor biopsies after drug treatment is an important objective of this study. Baseline biopsies will be performed following patient enrolling on study. If an initial attempt at percutaneous biopsy is unsuccessful, the patient will be given an option to proceed with a repeated attempt at percutaneous biopsy. A separate consent form must be signed for each biopsy procedure, so patients may choose not to undergo subsequent biopsies. If the baseline biopsy is unsuccessful or the patient refuses to undergo subsequent biopsies, no further biopsies will be performed, but the patient will remain on study, receive study medication, and other correlative studies will be performed. Similarly, patients who miss the first dose of drug will not subsequently be biopsied, but will remain on study.
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timing of tumor biopsies

For NR0B1 expression and gene expression signature tumor one tumor biopsy each will be obtained prior to treatment, and day +4 (+/- 1 day) during cycle 1 at steady state on treatment with mithramycin.
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biopsy procedure

No more than 2 tumor biopsy procedures per patient will be performed by Interventional Radiology. It is preferred that two core biopsies not less than 18-gauge in diameter and at least 1 cm in length are obtained. Only percutaneous biopsies will be performed. No biopsy of lung metastases will be attempted. It is estimated that there will be between 2-5 million cells from each biopsy. If a site is deemed appropriate for biopsy with minimal risk to the participant by agreement between the investigators and Interventional Radiology, an attempt at biopsy will be made. Determination of a disease site amenable to biopsy will be determined on an individual case basis after discussion with an interventional radiologist. The biopsy procedure to be used in this protocol is described below. Such biopsies can be safely performed as evidenced by literature reports (56) and Medical Oncology Branch experience at the Clinical Center. Risks of the procedure include, but are not limited to, bleeding, infection, pain, and scarring. We will follow Clinical Center Interventional Radiology SOPs for coagulant panel and platelets. All biopsies will be by percutaneous approach. No biopsy by an invasive (endoscopic, laparoscopic, or surgical) procedure will be performed. Only cutaneous, subcutaneous, or easily accessible parenchymal lesion core biopsies will be performed; however, there will be no core biopsies of lung lesions. The use of imaging to facilitate biopsies will be decided by members of the Interventional Radiology team and may include ultrasound, CT scan, or MRI. Should CT scan be needed for biopsy, the number of scans for each procedure will be limited to the minimum number needed to safely obtain a biopsy. All cases will be carefully reviewed with the interventional radiologists at NIH who have extensive experience in performing such procedures. Only if the procedure is considered to be low risk will we proceed with tumor biopsy in a given participant.
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biopsy processing

Tissue will be collected and immediately sectioned, a portion will be separated and frozen on dry ice for RNA collection for microarray and/or gene signature evaluation of EWS-FLI1 downstream target expression. Another portion will be imbedded in paraffin and sectioned. This tissue will be stained using immunohistochemistry for NR0B1 and if tissue remains, proteins in the apoptotic pathway will be evaluated including survivin (BIRC5), XIAP, Caspase-8 (CASP8),
and BCL-XL (BCL2L1). The remaining tissue will be distributed with the goal of establishing cell lines or performing basic laboratory investigations. Biopsy specimens will be collected by Dr. Brigitte Widemann and distributed to Dr. Helman's laboratory and the Laboratory of Pathology for analysis.  
Brigitte Widemann, MD NCI, POB 10 Center Drive, Building 10 CRC Room 1-5750, MSC 1101 Bethesda, MD 20892 Phone: 301-496-7387
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11.8 appendix 8 plasma samples

One mL of plasma will be obtained prior to initiating treatment with mithramycin on cycle 1 and prior to cycle 2 for future clinical reference in patients who experience bleeding, if needed. Obtain 2 mL of blood, separate plasma by centrifugation, and filter plasma through a 0.22 micron filter. Freeze the filtered plasma at -800C for future clinical reference if needed.  
Samples will be stored in PETS laboratory freezers. Plasma sample pre treatment with mithramycin cycle 1: Date obtained:________ Plasma sample pre cycle 2 of mithramycin: Date obtained:_________ Specific Instructions: Obtain blood in 6 ml purple top tube.
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spin at 4000 rpm x

OBTAIN FILTERED AND NON-FILTERED SAMPLES  
1) Non-Filtered sample - pipette out 1 ml from purple top tube and put into nuc, label as non-filtered, and freeze.  
2) Filtered samples -
a) pipette out greater than 1 ml from purple top tube and put into temporary nuc. b) Attach MILLEX GV 0.22 um filter to 5 cc syringe c) Attach large bore needle to exposed end of filter d) Extract 1 ml from nuc, discard temporary nuc e) Transfer from syringe (with filter and needle still attached) to a clean nuc, label as FILTERED, and freeze.
Supplies: Labels 3 1.8 ml nucs 5 ml syringe large bore needle Millex GV 0.22 um filter Pipettes Specimen bags 11.9 **APPENDIX 9:IMAGING STUDIES FOR VOLUMETRIC ANALYSIS OF TUMOR BURDEN** As a secondary endpoint, measurable disease will be quantified using volumetric MRI analysis. This analysis will be performed at the PETS laboratory. Imaging studies will be analyzed by Dr. Eva Dombi. :
Eva Dombi, MD NCI, POB 10 Center Drive, Building 10 CRC Room 1-5750, MSC 1101 Bethesda, MD 20892 Phone: 301-451-7023 11.10 APPENDIX 10: **PATIENT LOG FOR REACTIONS/SYMPTOMS** Patient ID: ____________________________ Cycle: ___________________ Dates of last Mithramycin infusion: _____________  
| Reaction/Symptom                                                                         | Start Date   | End Date   |
|------------------------------------------------------------------------------------------|--------------|------------|
| *Contact your study center team or local physician right away if you experience signs of |              |            |  
*Contact your study center team or local physician right away if you experience signs of bleeding. This may include, but is not limited to, petechiae (red or purple spots on the skin), nosebleeds; bruising; or blood in urine, stool or vomit. Patient/Parent signature: __________________ Date:______________________ Reviewed by:_____________________ RN/MD Date:______________________
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11.11appendix 11 patient log for

Patient ID:____________________________ Cycle: ____________________ Dates of last Mithramycin infusion: _____________  
| Medication Taken                                                                                        | Start Date   | End Date   |
|---------------------------------------------------------------------------------------------------------|--------------|------------|
| *Contact your study center team or local physician right away if you experience signs of bleeding. This |              |            |  
*Contact your study center team or local physician right away if you experience signs of bleeding. This may include, but is not limited to, petechiae (red or purple spots on the skin), nosebleeds; bruising; or blood in urine, stool or vomit.  
Patient/Parent signature: __________________ Date:______________________ Reviewed by:_____________________ RN/MD Date:______________________
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11.12appendix 12 monitoring toxicities between

Patient ID: _________________
Dates and Doses of last Mithramycin: ___________________ Cycle: _________
Dear Dr. ____________________, The patient named above is currently participating on a Phase I-II Mithramycin study conducted at the NCI, and requires the following lab tests and evaluations between ____________________ (date) and _____________________ (date)
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o history physical exam once

o **Weight**: Once Weekly o **Labs**: (Circle one) Twice Weekly during Cycles 1-3 or Once Weekly after Cycle 3.  
CBC/Diff Chemistry **Panel** *(include sodium, potassium, chloride, CO2, creatinine, glucose, BUN, albumin,* calcium, magnesium, LDH, phosphorus, uric acid)
LFT *(include alk phos, ALT/AST, Total bilirubin, Direct bilirubin, Total protein)*
Coagulation profiles - *ONLY If clinically indicated:**
Please FAX all lab results and medical summaries to: For any questions, please call: NAME OF STUDY CONTACT: Phone:
For urgent issues, contact the referring investigator at the following phone number:
(Physician Phone \# HERE)
***If the patient experiences any signs of bleeding (≤ Grade 2) including, epistaxis, hematuria,** hematemesis or blood in the stool, the patient will require immediate comprehensive clinical evaluation and coagulation profile (including PT/PTT/fibrinogen, thrombin time, PFA-100, Ddimer, Ca2+ **and ionized calcium)**
Please contact the number above with any clinically significant bleed and support with appropriate blood product support. Bleeding that does not stop within 6 hours with appropriate treatment will be considered a dose limiting toxicity and further dosing of mithramycin will be discontinued. _______________________________________ _____________________________________
Physician Signature Print Name Date:
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