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3.3 drug administration and treatment

Unless the patient already has a functioning permanent CVC, one will be placed prior to initiation of chemotherapy. Patients may receive up to three cycles of induction chemotherapy regardless of CD4 count at the time of study entry. The following tables are guidelines for schedules, rates and volumes, which, since these are standard induction chemotherapy regimens may vary slightly based on the clinical situation and guidance from personnel in the Pharmacy Department and clinical staff. 3.3.1.1 Induction Chemotherapy for lymphoma patients and some patients with leukemia:
EPOCH- Fludarabine may be administered as outpatient:  
| Version Date: 07/23/2018 Drug                            | Dose                                                                                                                      | Days                                                                                                                                                                                                                                                                                                                        |                |
|----------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|
| Fludarabine                                              | 25 mg/m2 per day IV                                                                                                       | Days 1,2,3                                                                                                                                                                                                                                                                                                                  |                |
| infusion over 30 minutes ± 10  minutes, daily for 3 days |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Etoposide                                                | 50 mg/m2 per day continuous IV                                                                                            | Days 1,2,3,4                                                                                                                                                                                                                                                                                                                |                |
| infusion over 24 hours ± 60  minutes, daily for 4 days   |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Doxorubicin                                              | 10 mg/m2 per day continuous IV                                                                                            | Days 1,2,3,4                                                                                                                                                                                                                                                                                                                |                |
| infusion over 24 hours ± 60  minutes, daily for 4 days   |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Vincristine                                              | 0.4 mg/m2 per day continuous IV                                                                                           | Days 1,2,3,4                                                                                                                                                                                                                                                                                                                |                |
| infusion over 24 hours ± 60  minutes, daily for 4 days   |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Cyclophosphamide                                         | 750 mg/m2 IV Infusion over 30                                                                                             | Day 5                                                                                                                                                                                                                                                                                                                       |                |
| minutes ± 10 minutes x 1 dose                            |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Prednisone                                               | 60 mg/m2 per day in 2-4 divided                                                                                           | Days 1,2,3,4,5                                                                                                                                                                                                                                                                                                              |                |
| doses PO daily for 5 days                                |                                                                                                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Filgrastim or                                            | Filgrastim: 5 Pg/kg per day SQ                                                                                            |                                                                                                                                                                                                                                                                                                                             |                |
| pegfilgrastim                                            | Filgrastim: Daily from day 6 until ANC  >1000/Pl x 2 consecutive CBC's                                                    |                                                                                                                                                                                                                                                                                                                             |                |
| Pegfilgrastim: 6 mg SQ, one  dose.                       | Pegfilgrastim: for patients t 40 kg, given 24-36 hours post chemotherapy (next cycle of chemo  therapy must be >14 days). |                                                                                                                                                                                                                                                                                                                             |                |
| 3.3.1.2                                                  | Induction chemotherapy for patients with leukemias (Non-myeloablative regimen):                                           |                                                                                                                                                                                                                                                                                                                             |                |
| Drug                                                     | Dose                                                                                                                      | Supportive Care                                                                                                                                                                                                                                                                                                             | Days           |
| Fludarabine                                              | 25 mg/m2 per day  IV infusion over  30 minutes ± 10  minutes, daily for  5 days.                                          | IV pre-hydration 1 hour prior to fludarabine daily  for 5 days with 0.9% NaCl and KCl 10 meq/liter  at a rate of 90 ml/m2 /hour to a maximum rate of  100 ml/hour. Following fludarabine infusion,  restart fluid at a rate of 90 ml/m2 /hour to a  maximum rate of 100 ml/hour continue until  cytarabine infusion begins. | Days 1,2,3,4,5 |
| Cytarabine*                                              | 2000 mg/m2 IV  infusion over 4  hours ± 30                                                                                | a. Corticosteroid ophthalmic drops 2 drops to  each eye every 6 hours starting prior to first  dose and until 24 hours after the last dose of  cytarabine completed.                                                                                                                                                        | Days 1,2,3,4,5 |  
56  
| Version Date: 07/23/2018 minutes, daily for                                                                                                                                                                                                                                                                                                                                                                  | b. Begin cytarabine doses 3.5 hours after                                    |                                                              |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|--------------------------------------------------------------|
| 5 days.                                                                                                                                                                                                                                                                                                                                                                                                      | completion of the preceding fludarabine.                                     |                                                              |
| b. Infuse in 250 ml of D5W.  c. IV post-hydration x 4 hours daily for 5 days  with 0.9% NaCl and KCl 10 meq/liter at a rate  of 90 ml/m2 /hour to a maximum rate of 100  ml/hour. d. In the event of signs of CNS toxicity,  cytarabine infusion will be interrupted and the  M.D. notified. No further cytarabine will be  administered if there is CNS toxicity (any  grade) deemed related to cytarabine. |                                                                              |                                                              |
| Filgrastim                                                                                                                                                                                                                                                                                                                                                                                                   | 5Pg/kg per day  SQ beginning the  day PRIOR to  initiation of  chemotherapy. | Daily from day  0 until ANC  >1000/Pl x 2  consecutive  days |  
3.3.1.3 **Subsequent Cycles**: Patients may receive subsequent cycles of chemotherapy as early as day 22 and up to 28 days after the previous cycle was initiated. Toxicities must resolve to < grade 2 in order to receive the next cycle with the following exceptions: liver function test elevations (if believed due to malignancy, manage as outlined in section **2.1.1.11**), neutropenia (ANC must be > 750/mm3 unless due to underlying disease in which case there is no grade restriction), lymphopenia, CD4 lymphopenia, leukopenia, and anemia (no grade restriction). An additional 14 days of recovery time is allowed before administration of subsequent cycles for resolution of toxicity or as medically indicated (e.g., documented infection).  
3.3.1.4 Dose Modification for Cycle 2 and Cycle 3
1.) Dose Reduction: If the prior cycle of induction chemotherapy is associated with reversible grade 4 non-hematologic toxicity, dose reduction for that patient's subsequent cycles will be performed. In addition, if 2 patients experience the same grade 4 nonhematologic toxicity, dose reduction will be performed for all subsequent cycles for all patients. Dose reduction will be as follows: a) EPOCH-F: Etoposide, doxorubicin, and vincristine will be reduced from 4 days to 3 days of administration and prednisone will be reduced from 5 days to 4 days. In the event of this change, cyclophosphamide will be given on day 4 and filgrastim will be started on day 5. The doses of all medications will remain unchanged.  
b) FLAG: Fludarabine and cytarabine will be reduced from 5 days to 4 days of administration. The doses of all medications will remain unchanged.  
3.3.1.5 Anti-emetics:
Routine anti-emetic prophylaxis and treatment should be employed. Corticosteroids may be used if clinically indicated. 3.3.1.6 Maximum dose of doxorubicin: As long as it has been determined that patients have adequate cardiac function as outlined in section **2.1.1.9**, doxorubicin will be administered until a maximum cumulative total dose of 500 mg/m2. Once this dose level has been reached, induction chemotherapy will not include doxorubicin. All other agents will be given according to the parameters outlined elsewhere in this section.
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3.3.2 transplantation

On day 22 after the final cycle of induction chemotherapy, patients will be eligible to start preHSCT preparative regimen. However, transplant may be postponed for up to an additional 14 days in order to facilitate cell processing, patient recovery from illness or other unanticipated delays. The following clinical parameters must be met in order to proceed to the transplant portion of this protocol: 3.3.2.1 Clinical Parameters Required for BMT:  
3.3.2.1.1 Non-hematologic toxicities of induction chemotherapy must resolve to < grade 2 in order to proceed. If more than 14 days of additional recovery time is necessary for resolution of toxicity or as medically indicated (e.g., documented infection), PI approval is required prior to proceeding to HSCT.  
3.3.2.1.2 ECOG performance status of 0,1 or 2RU.DUQRIV\RUIRUFKLOGUHQ\HDUVRI
DJH/DQVN\**Appendix 1**).  
3.3.2.1.3 Life expectancy > 3 months. 3.3.2.1.4 &DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$RU(&+2 IUDFWLRQDOVKRUWHQLQJE\(&+2 3.3.2.1.5 Renal function: age adjusted normal serum creatinine according to the table shown in 2.1.10 or a creatinine clearance of >60 ml/min/1.73 m2.  
3.3.2.1.6 /LYHUIXQFWLRQVHUXPGLUHFWELOLUXELQPJGOVHUXP$67DQG$/7[XSSHU
limit of normal.  
3.3.2.1.7 **Disease Status:** Patients must meet same disease criteria as enrollment as defined in section **2.1.1.1**
3.3.2.2 Inpatient care: Patients will be admitted for peri-transplant care to NIH in accordance with CC and nursing policies. Patients will be hospitalized in single positive pressure isolation rooms. 3.3.2.3 Pre-HSCT Non- Myeloablative Preparative Regimen: The following table is a guideline for schedules, rates and volumes, which, since these are standard pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and guidance from personnel in the Pharmacy Department and clinical staff. Pre-transplant chemotherapy will be administered as follows:  
| Drug        | Dose                         | Days                           |
|-------------|------------------------------|--------------------------------|
| Fludarabine | 30 mg/m2 per day IV infusion | Transplant Days –5, -4, -3, -2 |  
| over 30 minutes ± 30 minutes, daily for  4 days   |                                     |                                |
|---------------------------------------------------|-------------------------------------|--------------------------------|
| Cyclophosphamide                                  | 1200 mg/m2 per day IV infusion      | Transplant Days –5, -4, -3, -2 |
| Over 2 hours ± 30 minutes, daily for 4  days      |                                     |                                |
| Mesna*                                            | 1200 mg/m2 per day by continuous IV | Transplant Days –5, -4, -3, -2 |
| infusion, daily for 4 days.                       |                                     |                                |  
*Mesna will be administered at a dose of 1200 mg/m2 per day by continuous IV infusion on days
–5, -4, -3, and –2. The first 100 mg/m2 will be mixed with the cyclophosphamide in an appropriate amount of fluid based on the hydration rate. Immediately upon completion of this initial infusion, mesna at a dose of 1100 mg/m2 will be infused over 22 hours until the next cyclophosphamide dose or until 22 hours after completion of the final dose of cyclophosphamide. 3.3.2.4 Pre-HSCT Myeloablative Preparative Regimen for AML or subjects who have received prior TBI, as clinically indicated:
The following table is a guideline for schedules, rates and volumes, which, since these are standard pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and guidance from personnel in the Pharmacy Department and clinical staff.  
| Day      | Treatment                                                  |
|----------|------------------------------------------------------------|
| -6       | Anti Seizure Prophylaxis*                                  |
| -5 to -2 | Fludarabine 40 mg/m2 /dose IV daily (1.3 mg/kg if <10kg)** |
| -5 to -2 | Busulfan 3.2 mg/kg/dose IV daily x 4 doses***              |
| -1       | Rest (may be extended an additional day if needed)         |
| 0        | HSCT Infusion                                              |  
Note: Dose adjustments will be made for obesity, defined as > 125% of IBW:
Adjusted weight = 1.25*IBW (Please see **Appendix 13** for IBW and dose adjustment calculations.) * Seizure prophylaxis is mandatory. The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose, maximum dose: 2 mg) given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours after last busulfan dose. Alternative acceptable regimen is levetiracetam beginning 12 hours prior to busulfan and continue at least 24 hours after completion of busulfan. The dose of levetiracetam is 10 mg/kg/dose PO BID (maximum dose: 1000 mg). Loading dose and therapeutic monitoring are not necessary. For patients with a known seizure disorder, consider IV dosing with loading and therapeutic monitoring. NJ)OXGDUDELQHPJP,9GDLO\LQ1D&ORU':RYHUPLQXWHVRQ'D\V-5, - 4, -3, -2 for a total of 4 doses. < 10 kg: Fludarabine 1.33 mg/kg IV daily in 0.9% NaCl or D5W over 30 minutes on Days -5, -4, -3, -2 for a total of 4 doses. *** Busulfan administered intravenously via a central venous catheter as a 3-hour infusion once daily for 4 consecutive days for a total of 4 doses. Doses will be adjusted to achieve an overall exposure target AUC of 900-1500 (micromole/liter)*minute.
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3.3.2.5 pre-hsct myeloablative preparative regimen

The following table is a guideline for schedules, rates and volumes, which, since these are standard pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and guidance from personnel in the Pharmacy Department and clinical staff. Prep should be continuous.  
| Day                                                                        | Treatment*, **                                       |
|----------------------------------------------------------------------------|------------------------------------------------------|
| -6 to -4                                                                   | TBI 200 cGy BID (see Appendix 12 for TBI guidelines) |
| -3 to -2                                                                   | Cyclophosphamide 60 mg/kg/dose IV daily              |
| -3 to -2                                                                   | MESNA 60 mg/kg/dose IV daily                         |
| -1                                                                         | Rest (may be extended an additional day if needed)   |
| 0                                                                          | HSCT Infusion                                        |
| Note: Dose adjustments will be made for obesity, defined as > 125% of IBW: |                                                      |  
Adjusted weight = 1.25*IBW (Please see **Appendix 13** for for IBW and dose adjustment calculations.)
*The order of the cyclophosphamide/TBI may be switched to accommodate scheduling needs
**A CNS boost, if indicated, will precede the start of the conditioning regimen. Mesna infusion should start simultaneously with the cyclophosphamide and be administered by continuous IV infusion on days -3 and -2. The Mesna infusion should continue for a minimum of 22 - 24 hours after the last dose of cyclophosphamide. The entire Mesna dose prescribed should be administered each day of cyclophosphamide therapy.
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3.3.2.6 hydration during pre-hsct preparative

IV hydration will be initiated 12 hours prior to cyclophosphamide (on day –7 of the non myeloablative transplant, day -4 of the myeloablative ALL transplant) using 0.45% NaCl with 5% dextrose supplemented with KCl 10 meq/liter at a rate of 100 ml/m2/hour continuing until 24 hours after the last cyclophosphamide dose has been completed. During hydration, IV furosemide (0.5 mg/kg/dose to a maximum of 20 mg) may be administered as needed to maintain normal urine output and fluid balance. During hydration, serum electrolyte levels will be monitored every 12 hours and IV fluid content and KCl supplementation will be adjusted to maintain normal serum electrolyte levels. Hydration volume, composition and/or rate may be modified based on patient condition and investigator assessment, but may not go below the minimum of 65 ml/m2/hour.  
3.3.2.7 GVHD Prophylaxis with calcineurin inhibitor: 3.3.2.8 Anti-emetics: Routine anti-emetic prophylaxis and treatment should be employed. Corticosteroids may be used if clinically indicated. 3.3.2.9 Stem Cell Infusion: On day 0, the patient will receive fresh donor CD34+ PBSCs via intravenous infusion. (Cryopreserved cells may also be used in clinically indicated.) If the stem cells are cryopreserved, the product will be thawed and administered IV immediately at a rate of approximately 10-15 ml/min or as tolerated based on volume status and/or DMSO toxicity. Do not exceed 20 ml of product per kg body weight (if 10% DMSO) or 40 ml of product per kg body weight (if 5% DMSO) so that per 24-hour period so that total DMSO administered is less that 1 mL/kg/day.  
x Tacrolimus will be initiated on the day -1 (at least 24 hours before the stem cell infusion)
typically starting at a dose of 0.02 - 0.03 mg/kg/day as a continuous infusion. Once oral feedings are tolerated and there is resolution of mucositis, parenteral tacrolimus will be discontinued. Twelve hours later, oral tacrolimus will be initiated at a dose of 0.1-0.15 mg/kg/day in two divided doses every 12 hours.  
x The tacrolimus dose will be titrated to maintain a trough level of 5-10 ng/ml. Tacrolimus levels should NOT be drawn from the CVL lumen used for tacrolimus infusion.  
OR
x Cyclosporine-A will be initiated on the day before the transplant (day –1) starting at a dose of 2 mg/kg/dose IV over 2-hours q 12 hours.  
x The dose will be titrated to maintain an approximately trough level of 200-250 ng/ml
(monoclonal RIA methodology). CSA levels should not be drawn from the CVL lumen used for CSA infusion.  
x Once the patient is able to tolerate oral feedings, CSA will be converted to an equivalent oral dose. The starting oral dose should be approximately 2-times the IV dose given in a q 12 hour schedule. The dose should be titrated to a trough level of 200-250 ng/ml (monoclonal RIA methodology).  
x Tacrolimus is preferred over cyclosporine-A, however if tacrolimus is unavailable, or the laboratory reagents needed to monitor tacrolimus serum levels are unavailable, cyclosporine-A may be substituted, x Calcineurin inhibitor will continue until day 100 post-transplant, at which point it will be gradually tapered as long as the level of acute-GVHD is < grade 2 and there is no chronicGVHD. Taper will consist of a 5 to 10% dose reduction each week and will be discontinued by around day 180 post-transplant.  
x The decision to taper Caclineurin inhibitor before day 100 will only be permitted if clinically indicated and after PI approval. Specifically, taper before day 100 may be permitted for the treatment of progressive disease and/or low levels of donor chimerism.  
The target dose of the PBSC is > 5 x 106 CD34+ cells per kg recipient body weight.  
a. Pre-medications: Patients will receive the following medications approximately 30-60 minutes prior to stem cell infusion:
i. Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV
ii. Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO
iii. If the patient has been premedicated earlier in the day for blood product or other infusion, pre-medication is not warranted if given within 4 hours of stem cell infusion.  
b. Hydration During Stem Cell Infusion: IV hydration will be initiated at a minimum of 12 hours prior to stem cell infusion (on day –1) using 0.9% NaCl supplemented with KCl 10 meq/liter. Patients will receive hydration at a rate of 90 ml/m2/hour to a maximum rate of 100 ml/hour. Hydration will continue at a minimum of 12 hours after the stem cell infusion has been completed.  
c. Monitoring During Stem Cell Infusion (Inclusive of T-cell Addback as a separate infusion): General monitoring guidelines will include vital signs and oxygen saturation q 15 minutes (+/- 10 minutes) until one hour after completion of infusion (s) and will follow NIH Clnical Center Nursing and Patient Care Services Procedure "Administration of products for Cellular Therapy." (http://intranet.cc.nih.gov/nursing/practicedocs/procedures_pdf/PRO_Cellular_Therapy_I nfusion.pdf)
d. Supplemental oxygen will be available at the bedside. e. If an allergic or other acute reaction occurs, studies appropriate for investigation of a transfusion reaction will be performed (urinalysis, CBC, Coomb's test).  
3.3.2.10 Growth Factor Administration Post-transplant:
After stem cell infusion on day 0, filgrastim will be started at a dose of 5Pg/kg/day SQ, unless contraindicated. Filgrastim will continue until the ANC is greater than 5000 cells/Pl (post nadir)
on two consecutive blood draws. If the patient has impaired renal function, pegfilgrastim may be administered in place of filgrastim at 6 mg RQHWLPHGRVHIRUSDWLHQWVNJRQGD\
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3.3.2.11 nk cell infusion

On day 21 (+/- 3 days) after stem cell infusion, the dose of NK cells will be infused intravenously over approximately 10 -20 minutes. In the absence of DLT, the NK cell infusion will be repeated 28 (+/- 7 days) days following the first infusion for a total of 2 infusions, as per the dose descriptions in Section 3.1. Any adverse reaction should prompt interruption of the infusion and should be reported to the principal investigator. Patients will receive the following medications approximately 30-60 minutes prior to NK cell infusion:
1.) Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV; 2.) Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO 3.) If the patient has been premedicated earlier in the day for blood product or other infusion, pre-medication is not warranted if given within 4 hours of stem cell infusion Safety monitoring during and after infusion will follow NIH clnical center guidelines (http://intranet.cc.nih.gov/nursing/practicedocs/procedures_pdf/PRO_Cellular_Therapy_Infusion .pdf) and will include vital signs (prior to infusion, every 15 minutes (+/- 10 minutes) for 1 hour
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62

after the start of infusion, every 30 minutes (+/- 15 minutes) for the second hour and then hourly until stable. An NK-DLI infusion may be delayed in patients with significant complications (eg. severe infection, engraftment syndrome, GVHD flare) that according to the protocol PI or AI, may be in the best interest of the patient, and later administered when the patient has sufficiently recovered. Cells must meet the same release criteria as the originally scheduled dose regimen. For statistical analysis, patients receiving infusions outside of the planned ± 3 day or 7 day time frame will be analyzed separately and reported descriptively. NK cells may remain in culture for up to an additional 7-14 days. If both doses of NK cells can be delivered within the prescribed time, this will be considered feasible delivery of NK-DLI. If a DLT occurs, no further NK cells will be given to that patient.
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3.4 on study protocol evaluation

3.4.1 Baseline  
3.4.1.1 Spinal fluid for cell count and cytology (for patients with acute leukemia or NHL). 3.4.1.2 PCR of mini-satellite regions (STR profile) for future determination of chimerism
(donor and patient). These studies may be performed at any time prior to entry without time limitation.  
3.4.1.3 Antibody screen for varicella, HSV, and toxoplasma (patient only) and CMV, EBV
(patient and donor).  
3.4.1.4 PPD with appropriate control (for patients considered to be at high risk). 3.4.1.5 CBC with differential (patient and donor). Must be repeated within 72 hours of first collection and within 24 hours of subsequent collections (donor).  
3.4.1.6 TBNK (patient only) 3.4.1.7 Urinalysis, (patient and donor). 3.4.1.8 Lupus anti-coagulant (patient only), as indicated.  
3.4.1.9 Serum iron, ferritin and transferrin (patient only).  
3.4.1.10 24-hour urine for creatinine clearance, if indicated. 3.4.1.11 Spot urine for calcium/creatinine ratio (patient only). 3.4.1.12 Radiologic studies that measure identifiable disease may be repeated prior to each cycle of immune depleting chemotherapy and prior to the conditioning regimen, at the PI's discretion.  
3.4.2 Induction Chemotherapy Cycles (EPOCH-F or FLAG cycles)
3.4.2.1 H&P prior to each cycle and weekly during induction.  
3.4.2.2 CBC, differential, platelets and prior to each cycle and two times per week during induction.  
3.4.2.3 Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid,- prior to each cycle and one time per week during induction.  
3.4.2.4 Urinalysis prior to each cycle. 3.4.2.5 Urine or Serum Pregnancy test prior to each cycle, for all females of childbearing potential.  
3.4.2.6 Disease staging evaluation - as clinically indicated based on disease and sites of disease
(see section 2.2).
3.4.2.7 TBNK or NK panel (CD4 count) within 7 days prior to each cycle. 3.4.3 Pre-HSCT evaluation 3.4.3.1 H&P  
3.4.3.2 CBC, differential, platelets 3.4.3.3 Laboratory evaluations: *Sodium (Na), Potassium (K), Chloride (Cl), Total CO2*
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid 3.4.3.4 Urinalysis
3.4.3.5 Complete disease staging evaluation - staging based on disease and sites of disease within 17 days prior to start of transplant preparative regimen.  
3.4.3.6 EKG and MUGA or ECHO
3.4.3.7 Research studies (Section 3.5, **Appendix 4**) (When ALC > 200 K/uL ONLY)  
a. Lymphocyte Phenotyping:
- TBNK (CD4 count) or - NK Subset (except on Fridays) within 7 days prior to each cycle (See **Appendix 4**).  
3.4.4 HSCT Hospitalization
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3.4.4.1 h&p daily

a. Twice daily weights (from transplant through date of engraftment)
b. DAT daily on days 6-10 (for patients with ABO histo-incompatabilities ONLY)  
3.4.4.2 CBC, differential, platelets daily. 3.4.4.3 Laboratory evaluations: *Sodium (Na), Potassium (K), Chloride (Cl), Total CO2*
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT,
AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid daily.  
3.4.4.4 Type and screen every 4 days (as clinically indicated) 3.4.4.5 Reticulocyte count, haptoglobin, IGG level, EBV, CMV, HHV6, adenovirus monitoring weekly (during hospitalization).  
3.4.4.6 Urinalysis daily
3.4.4.7 Research studies (Section 3.5, **Appendix 4**)  
3.4.4.8 Disease staging evaluation (as clinically indicated based on disease sites) 3.4.5 Evaluations After HSCT discharge through day 100 (±30 days), as clinically indicated 3.4.5.1 H&P twice weekly 3.4.5.2 CBC, differential, platelets weekly. 3.4.5.3 EBV, CMV, HHV6, adenovirus monitoring weekly.
3.4.5.4 Type and screen and IGG level, weekly as clinically indicated.  
3.4.5.5 Laboratory evaluations: *Sodium (Na), Potassium (K), Chloride (Cl), Total CO2*
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid weekly.  
3.4.5.6 Iron studies - serum iron, transferrin, ferritin, reticulocyte count, monthly as clinically indicated.  
3.4.5.7 Bone marrow biopsy and aspirate day 28 (as indicated).  
3.4.5.8 Research studies (Section 3.5, **Appendix 4**).  
3.4.5.9 Blood for bcr-abl PCR 28-35 days post final NK cell infusion.
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3.4.6 evaluations after day 100

Disease evaluations may occur before Day 100 or at any time after day 100 based on signs and symptoms or PI's suspicion of disease recurrence. Follow-up visits will be clinically determined after day 100, but at least once monthly. NIH scheduled visits will occur around day 100, day 150, day 180, day 270, and day 365 post-transplant. These timepoints are used a guidelines but may be altered based on each patient's clinical condition. Patients will then be followed approximately every 3 months for the second post-transplant year, and then every 6 months for the third post-transplant year, and then yearly until at least 3 years post-transplant. Evaluations will include the following as clinically indicated:  
3.4.6.1 H&P including symptoms of GVHD (see Appendices 6-8), infection, and respiratory dysfunction.  
3.4.6.2 CBC and reticulocyte count 3.4.6.3 Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total,
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid 3.4.6.4 IgG levels monthly until IVIG supplementation discontinued. 3.4.6.5 Blood type, red cell phenotype, and ABO isohemagglutinin titers (in cases of ABO
incompatibility only) as clinically indicated.  
3.4.6.6 Antibody titers to previous vaccinations or infections (e.g., diphtheria, HIB, HBSAb, pneumococcus, tetanus, varicella) at or beyond 12 months post-HSCT and off IVIG for at least 3 months.  
3.4.6.7 Disease staging evaluation (as clinically indicated based on disease sites): All studies that identify disease will be performed around day 100, every 3 months for the first year, every 6 months for the second and third year, and yearly for years 4-5, or as clinically indicated.
3.4.6.8 Organ function evaluation:  
a. Pulmonary function tests with DLCO q 3 months if GVHD, otherwise every 6 - 12 months and as clinically indicated.  
b. Cardiac tests: EKG and MUGA or ECHO at around 6 months, 1 year, and then yearly until year 5 ± 3-4 months.  
c. Endocrine studies as clinically indicated.  
3.4.6.9 Research studies (Section 3.5, **Appendix 4**) including KIR/HLA
genotyping/immunogenetics at or around Day 100±30 days.  
3.4.7 Related Donor Evaluations During Treatment (**Appendix 2 B**)
3.4.7.1 Donors will have an interim health assessment performed by DTM staff immediately before each collection procedure.  
3.4.7.2 Infectious disease testing will be performed no more than 30 days prior to each collection.  
3.4.7.3 If a CVC is required, CBC, PT, PTT, and history and physical must be performed within 72 hours prior to placement.
3.4.7.4 Donors will have a physical examination, including a CBC, in the ETIB or POB
clinic or by their primary physician within one week of each collection.
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3.5 nci/nih biologic studies

Blood sample volume for the research purposes of this study will be restricted. The amount of blood drawn from adults (those 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, in an 8 week period. The amount of blood drawn for research purposes from pediatric patient subjects (those under 18 years of age) will not exceed 5 mL/kg in a single day, and no more than 9.5 mL/kg over an 8 week period. In the event that blood draws are limited due to these restrictions, research studies will be performed in order of priority as listed below and in **Appendix 4**.  
3.5.1 Proposal for Immune Function Studies (Brown)  
3.5.1.1 Background: to evaluate the effects of the chemotherapy regimen on host immune depletion/reconstitution.  
3.5.1.2 Rationale: Analysis will consist of flow cytometry to detect depletion of lymphoid subpopulations during therapy.  
3.5.1.3 Objectives / Specific Aims: 3.5.1.3.1 Immunophenotyping
Blood samples (5ml lavender top tube) will be drawn Samples should be delivered to the Clinical Pathology Flow Cytometry Laboratory, Attn. Margaret Brown, Building 10, Room 2C410, 301-496-4879. Test must be scheduled prior to sending.  
Time points for Lymphocyte Phenotyping–NK Subset samples will be: 1) On-study; post-HSCT
at 24 hours (+/- 24 hours) post NK\#1 and \#2 infusion (+/- 48 hours) and on day 28 (+/- 7), day 60 (+/- 21), and day 100 (+/- 30), and then every 3 months (+/- 1 mo) for the first two years posttransplant. Samples will not be drawn if ALC is < 200 K/uL.  
Time points for Lymphocyte Phenotyping TBNK samples will be: 1) Prior to cycle 2 and 3 of induction chemotherapy (within 7 days of each cycle); 2) Prior to pre-HSCT preparative chemotherapy, (day –7), and 3) Prior to HSCT (day 0) if ALC is > 200 K/uL. Samples will not be drawn if ALC is < 200 K/uLSamples should be delivered to the Clinical Pathology Flow Cytometry Laboratory, Attn. Margaret Brown, Building 10, Room 2C410, 301-496-4879. 3.5.2 Chimerism Analysis (Kurlander) Blood samples (10cc and, if doing cell sorting, an additional 20* cc in yellow top ACD tubes) will be drawn to evaluate the relative percentage of donor and host chimerism post-transplant. Samples should be delivered to the clinical laboratory of Dr. Roger Kurlander in Building 10, hematology laboratory (301-435-6399) * Cell sorting will be done on day 14 and repeated on day 28 if mixed chimerism (<98% donor) is found on day 14. Timepoints for samples will be: 1) Eligibility; 2) Day 14 (+/- 5), day 28(+/- 7), day 60(+/- 21), day 100(+/- 30) ; and 2) After day 100 as clinically indicated. 3.5.3 Serum Cytokine Analysis (Lab) Blood samples (5 cc in red-top serum collection tubes) will be drawn to evaluate circulating cytokine levels (including IL7 and IL15) and possible correlation with the degree of CD4 lymphopenia, immune reconstitution, engraftment, and clinical complications such as graft versus host disease and infections. The serum will be separated and frozen for subsequent analysis by high sensitivity colorimetric enzyme linked immunosorbant assay. Timepoints for samples will be: 1) On-Study; 2) Prior to pre-HSCT preparative chemotherapy (day –7); 3) Prior to HSCT (day 0); and 4) day prior to or morning of each NK infusion (before infusion) then day 1, 2, 7 (+/- 48 hr) and 14 (+/- 48 hr) days post each NK infusion, then days 60
(+/- 21) and 100(+/- 30), and then every 3 months (+/- 1 mo) for the first two years posttransplant.  
Samples should be picked up by courier (301-846-5893) and delivered to Frederick Repository. 3.5.4 TH1/TH2 Cytokine Profile (Frederick Repository) Blood samples (1 cc in red-top serum collection tubes) will be drawn to evaluate the types of cytokines that are present in the allogeneic environment, and correlate their levels with occurrence of potential GVHD and infectious complications. Blood samples will be analyzed by multiplex cytokine bead array technology. Timepoints for samples will be: 1) On-Study; 2) Prior to pre-HSCT preparative chemotherapy (day –7); 3) Prior to HSCT (day 0); and 4) and post-HSCT on day 1, day prior to or morning of each NK infusion (before infusion) then day 1, 2, 7 (+/- 48 hr) and 14 days (+/- 48 hr) post each NK infusion, then days 60 (+/- 21) and 100 (+/- 30), and then every 3 months (+/- 1 mo) for the first two years post-transplant. Samples should be picked up by courier (301-846-5893) and delivered to Frederick Repository. 3.5.5 KIR/HLA genotyping/immunogenetics (Carolyn Hurley - Georgetown Univ.) complete  
3.5.5.1 Objective: Retrospectively evaluate for the presence or absence of specific KIR genes to determine if the presence of a specific KIR in the absence of its HLA ligand (ie KIR "mismatch") correlates with enhanced engraftment, increased anti-tumor activity and less infectious complications.  
3.5.5.2 Rationale: Patients receiving KIR mismatched NK-DLI may have better NK activity due to the absence of inhibitory signals, but it is not clear if KIR mismatch is needed in the setting of multiple activating signals provided during the NK cell expansion process.  
3.5.5.3 Methods: Presence or absence of specific KIR genes will be determined by locus specific PCR amplification followed by gel electrophoresis. The panel of primer pairs will detect KIR2DL1-2DL5, 2DS1-2DS5, 3DL1-3DL3, 3DS1 and two pseudogenes. Followup sequencing of KIR alleles may be performed if required to identify expressed alleles. Timepoint for samples will be prior to SCT (day 0) and day 100. Samples (1-2 ml blood in ACD or 3 buccal swabs will be kept at room temperature and will picked up by the research nurse. They will then be shipped deidentified to Lihua Hou / Carolyn Hurley at C. W. Bill Young Marrow Program/Georgetown University, 3rd Floor, 11333 Woodglen Drive, Rockville, MD 20852, 301-998-8900.  
3.5.6 Viral monitoring for functional Immune Status 3.5.6.1 Objective: Serially monitor viral reactivation in transplant patients. Compare viral reactivation to historic controls on protocol 02-C-0259 and 01-C-0125. Monitor for a meaningful correlation between circulating NK cell levels and viral reactivation.  
3.5.6.2 Rationale: Patients receiving TCD grafts carry a higher risk for viral infections and reactivation. Viral reactivation is a marker for T-cell immune dysregulation. In particular, HHV6 is a marker of cell mediated viral response and a marker of inflammation.  
3.5.6.3 Methods: Weekly PCR surveillance of EBV, CMV, HHV6, and adenovirus through the Clinical Center central laboratory, Section of Microbiology.  
3.5.7 Research Testing of Biopsy Samples 3.5.7.1 Xenograft Models: Human tumors can be transferred to immunodeficient mice. These xenografts models can be used to study tumor biology and treatment. Samples from consenting adult patients with solid tumor malignancies will be used for pre-clinical testing and development of immunotherapy strategies. Recent studies exemplify the important role for xenograft models in the pre-clinical development of IGFR-1 antibodies. Informed consent will be required for this purpose, and subjects will be given the option to decline having xenograft models established from their samples.
Sample requirements: Fresh tumor samples in DMEN + 10% FBS, (optimally 0.5 cm in one dimension). Samples will be minced into 1mm fragments and injected into immunodeficient mice in a location appropriate for specific tumor growth (adrenal gland for neuroblastoma, bone for osteosarcoma). Must schedule with lab in advance.  
3.5.7.2 Cell line establishment: Attempts will be made to establish solid tumor cell lines lines with the aim to identify potential molecular targets for new therapy development. Cell lines may also be used to perform in vitro testing of new agents. Informed consent will be required for this purpose, and subjects will be given the option to decline having cell lines established from their samples.
Sample requirements: Fresh tumor samples in DMEN + 10% FBS, (optimally 0.5 cm in one dimension). Samples will be minced into 1mm fragments and cells will be placed in flasks with complete growth media and incubated in humidified tissue culture incubator.  
3.5.8 NIH Policy on Research Use of Stored Human Samples, Specimens, or Data: Blood and tissue collected during the course of this study will follow NIH guidelines for the research use of human samples. Most samples will initially be sent to the Central Repository for NCI/Frederick using established procedures. All samples will be stored in monitored freezers/refrigerators either in the Central Repository for NCI/Frederick or in the investigator's laboratory at specified temperatures with alarm systems in place. All samples will be tracked by unique sample identifiers. A secure computer database system will be used to track all samples collected on this protocol; the system will contain data that includes but is not limited to the unique sample identifiers, storage locations and conditions, biologic study results, information about the sample origins (as appropriate), and corresponding records of all derivatives generated from samples collected on this protocol. The system will employ mechanisms for restricting users to viewing only the level of data appropriate for each individual user, will provide the capability to audit any data modification, and will be maintained and backed up according to established standards. Specimens will be disposed of in accordance with the environmental protection laws, regulations and guidelines of the Federal Government and the State of Maryland. Any new uses of human subject samples collected during the course of this trial must be reviewed and approved by the NCI IRB. Any loss or unintentional destruction of the samples will be reported to the IRB.
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3.6 concurrent therapy 3.6.1 treatment

In the event a patient develops GVHD, the subsequent procedures will be followed. Standard clinical and/or biopsy criteria will be used to establish the diagnosis of GVHD, attempts will be made to obtain histiologic confirmation. Acute GVHD (aGVHD) will be graded by the CIBMTR
criteria (**Appendix 5**) and Chronic GVHD (cGVHD) by the NIH cGVHD Consortium Consensus Criteria (**Appendix 6** and **Appendix 7**). Patients will be monitored for GVHD over the entire 5-year post-HSCT observational period. Patients with grade 1 acute GVHD will not receive systemic therapy. In general, patients with > grade 2 acute GVHD will be treated with corticosteroids. Patients who fail to respond satisfactorily to corticosteroids will be considered for other standard anti-GVHD therapies, and/or investigational GVHD treatment protocols if available. Simiarly, patients with cGVHD will be treated with corticosteroids and/or calcineurin inhibitors. Patients will also be considered for other standard anti-GVHD therapies, and/or investigational GVHD treatment protocols if available. Established guidelines, which are regularly reviewed and updated for the treatment of GVHD as outlined by the NIH BMT consortium will be followed. These guidelines can be found at http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml.  
3.6.2 Immunosuppressants Calcineurin inhibitor as described in section **3.3.2.6**.  
3.6.3 Infection Prophylaxis Infectious prophylaxis as outlined in section 4.1.  
3.6.4 GI Protective Agents Patients on corticosteroids must be on H2 blockade or other gastrointestinal protective agents. 3.6.5 Hematologic and Blood Product Support 3.6.5.1 Platelet transfusion: In attempt to prevent bleeding, platelet counts should be maintained
> 20,000/ul (or as clinically indicated) and >50,000 prior to apheresis. Single donor platelets should be employed whenever possible in attempt to decrease donor exposure and the risk of alloimmunization.  
3.6.5.2 Red blood cell transfusion: Packed RBCs should be used to maintain a hemoglobin > 8 gm/dL for ABO compatible transplants and >10 gm/dL in ABO minor mismatch transplants (or as clinically indicated).  
3.6.5.3 Irradiation: To reduce the risk of transfusion associated graft-vs-host disease, all cellular blood products must be irradiated.  
3.6.5.4 Leukodepletion: To decrease the risk of febrile reactions and alloimmunization, platelets and red cells should be leukoreduced.
3.6.6 Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma: Concurrent therapy or prophylaxis for testicular leukemia, CNS leukemia, and CNS lymphoma including standard intrathecal chemotherapy and/or radiation therapy will be allowed as clinically indicated after approval by the PI. Such treatment may continue until the planned course is completed. Patients must be in CNS remission at the time of protocol enrollment.  
3.6.6.1 CNS Prophylaxis for Patients with ALL: Patients with ALL may receive intrathecal (IT)
methotrexate for 5 doses after transplant.
a. Dose: By age as follows:  
| Age (years)   | Methotrexate (mg)*   | Volume (ml)   |
|---------------|----------------------|---------------|
| 3-8           | 12                   | 8             |
| >9            | 15                   | 10            |  
b. Administration: To facilitate CNS delivery, the volume of CSF removed should be approximate the volume administered and patients should remain prone for 30 minutes after IT administration. c. Schedule: Approximately weekly x 5 doses to begin after myeloid recovery, not before post-transplant day 28. d. Antiemesis: Routine (e.g., ondansetron) should be considered prior to each dose of methotrexate in patients with a history of nausea associated with intrathecal chemotherapy. *Methotrexate may be substituted with hydrocortisone/cytarabine, or Triple IT (MTX/AraC/HC) may be given, if clinically indicated, and dosing will be performed as per standard Children's Oncology Group (COG) doses (example below):  
| Age (years)   | Cytarabine (mg)   | Hydrocortisone (mg)   |
|---------------|-------------------|-----------------------|
| 3-8           | 24                | 12                    |
| >9            | 30                | 15                    |
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3.6.7 treatment of progressive disease

Patients who have progressive disease after day +100 post-transplant may receive donor lymphocyte infusions (DLI) since this has been effective in the management of post-BMT
relapse. Patients who have been started on immunosuppression for GVHD treatment will undergo sequential withdrawal of immunosuppression prior to DLI. As long as there is no evidence for active GVHD, patients may be eligible to receive unmodified DLI. If deemed necessary to combat rapidly progressive disease, adjunctive chemotherapy using standard disease-specific agents may be employed in conjunction with this approach with PI approval. The specific chemotherapy to be used should be decided by the patient's oncology and BMT teams in consultation with the PI. Lympholytic agents (e.g., corticosteroids) should be avoided. Alternatively, in the event of progressive disease after day +100, the patient may be taken off-study and referred for conventional therapy or participation on other NCI investigational trials for which they might be eligible.  
3.6.7.1 **LEUKAPHERESIS PROCEDURE TO COLLECT CELLS FOR DLI**: Donation of DLI shall be by standard lymphapheresis, during which 2 to 5 blood volumes will be processed as estimated by recipient weight and target cell harvest dose (maximum 5 total blood volumes) in children (defined as less than 40 kg), or 7 to 10 liters of blood in adults will be processed, depending on recipient weight (volume processed to be determined by DTM staff in consultation with PI). Non-mobilized donor lymphocytes will be collected by apheresis and processed as per standard NIH DTM procedures. The lymphapheresis will undergo red cell or plasma depletion for major or minor ABO incompatibility, respectively, according to CPS, DTM policies and procedures. In the event that some or all of the donor lymphocytes will be administered in the future, the lymphapheresis product will be cryopreserved and stored according to CPS, DTM standard operating procedures. 10% of the total lymphapheresis product of related donor ONLY will be cryopreserved as a separate aliquot and stored for research. Use of cryopreserved DLI collected as part of the original PBSCT collection may be used if clinically indicated.  
3.6.7.2 **DLI ADMINISTRATION**: DLI will generally be administered at a starting dose of 1 x 106 CD3+ T-cells/kg recipient body weight by IV infusion. A higher starting may be used as clinicaly indicated. Additional DLI may be administered in escalating doses in increments of 0.5-1 log10 to a maximum dose of 1 x 108 CD3+ T-cells/kg every 2-4 weeks provided there has been no improvement in malignant disease and there remains no evidence of GVHD.  
Patients will receive the following medications approximately 30-60 minutes prior to DLI cell infusion:
1.) Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV or PO over 10-15 minutes; 2.) Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO 3.) If the patient has been premedicated earlier in the day for blood product or other infusion, pre-medication is not warranted if given within 4 hours of stem cell infusion Safety monitoring during and after infusion will include vital signs (blood pressure, heart rate, respiratory rate) prior to infusion, and 15 minutes post infusion and more frequently if clinically indicated.
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3.6.8 treatment of mixed chimerism

If a patient has < 95% donor lymphoid chimerism on day +28 post-transplant, repeat analysis will be performed until complete chimerism is achieved or a pattern of stable mixed chimerism or graft rejection is established. For patients with significant reductions in donor chimerism as determined by the PI, DLI (as per Section **3.6.7**) will be considered. If patient is on immunosuppression for GVHD) sequential withdrawal of immunosuppression will precede DLI administration. This approach has been successfully employed in pediatric patients with ALL, AML, and MDS who had progressive mixed chimerism after conventional myeloablative allogeneic BMT(212). If at any time a patient has < 5% donor lymphoid chimerism, they will be taken off treatment and consideration will be given for conventional HSCT.  
3.6.9 Treatment of Graft Failure/Graft Rejection:
If a patient develops graft rejection (as evident by decreasing hematopoiesis and decreasing CD3 donor chimerism) or has graft failure (lack of engraftment) they will be taken off treatment and consideration will be given for conventional HSCT on-study. Therapy for the treatment of graft rejection will be determined as clinically indicated but may include serotherapy such as anti-thymocyte globulin, alemtuzumab and/or second transplant.
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3.7 criteria for removal from

3.7.1 Criteria for removal from protocol therapy x Unacceptable toxicity as defined in section **3.1.1**.  
x Inability to proceed with transplantation (failure to meet criteria). x Progressive disease during induction therapy. x Irreversible non-hematologic grade 4 toxicity during induction chemotherapy. Any other non-hematologic toxicity during induction chemotherapy of sufficient severity or duration that in the opinion of the PI poses prohibitive risks to further chemotherapy.  
x Progressive disease post tranplant, which requires anti-neoplastic therapy.  
x Patients unable to comply with study requirements will come off treatment.  
x < 5% donor lymphoid chimerism at or beyond day +28, or graft rejection/failure x Enrollment on another clinical trial for treatment of progressive disease or graft rejection.  
x Recipients will come off treatment upon completion of study follow-up period (5 year post PBSCT). These patients with be followed for survival and development of chronic GVHD.  
3.7.2 Off-Study Criteria x Recipient death. x Recipient withdrawal of consent x Lost to follow-up x Investigator discretion x Completed study follow-up period x Donors will come off-study at the time of recipient off-study date Note: all minors who sign the treatment consent will be kept on study and followed until the age of 18 to allow for consent. 3.7.3 Off Protocol Therapy and Off-Study Procedure Authorized staff must notify Central Registration Office (CRO) when a subject is taken off protocol therapy and when a subject is taken off-study. A Participant Status Updates Form from the website (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov.
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3.8 end of study evaluation

The following tests and procedures should be performed, if possible, at the time a patient comes off study regardless of the reason for coming off study, unless the test or procedure has been performed in the last 4 weeks (2 weeks for physical examination and laboratory assessment). 3.8.1 History and physical examination. 3.8.2 Laboratory Assessment: Iron studies (serum iron, ferritin, and transferrin), Complete blood count, differential and platelet count. Chem 20 and urinalysis.  
3.8.3 Disease restaging 3.8.4 Apheresis. (Optional)
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3.9 duration of study

Patients will be followed annually for up to year 3 after completing treatment on study, and annually by phone for survival status.
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4 supportive care 4.1 infection

Routine measures to prevent infection in accordance with standard of care practice. For patients enrolled at the NIH, the recommendations following the NIH Allogeneic Stem Cell Transplantation guidelines should be employed (http://intranet.cc.nih.gov/bmt/education/infectious-mgmt-guidelines.shtml).
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4.2 mucositis prophylaxis

Kepivance™ (palifermin), a human keratinocyte growth factor (KGF), may be administered to reduce the incidence or severity of mucositis. Palifermin will be administered at a dose of 60 mcg/kg/day IV X 3 doses, given twice. The first 3 daily doses should be administered prior to the preparative chemotherapy with the 3rd dose completed at least 24 hours prior to starting the preparative regimen. The last 3 daily doses should be administered starting on day +1.
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5 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant.  
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days following the last dose of study drug. AEs that are considered treatment related, expected, continuing, but not resolvable by 30 days after treatment completion (e.g., alopecia) will not be followed after the 30-day period. Donor AE data will be collected through 1 week following collection. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
x Results in discontinuation from the study  
x Is associated with clinical signs or symptoms x Requires treatment or any other therapeutic intervention x Is associated with death or another serious adverse event, including hospitalization.
x 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. Recipient and Donor data will also be reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) registry as required by law. Recipients and donors will be offered the opportunity to consent to the use of this information, and additional baseline and outcome data submission, through one of two NIH protocols, depending on their donor source. Consent to these protocols is optional and does not affect the recipient's access to transplant or to unrelated donor grafts. For unrelated donor graft recipients, this data submission element ensures compliance with standards described in the Transplant Center Agreement between the NIH Clinical Center and the NMDP.  
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the IRB will be notified.
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5.2 response criteria

Standard disease specific clinical and laboratory response criteria will be employed as detailed in Appendix 9 and **Appendix 10**.
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5.3 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40).
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5.4 data reporting exceptions

For patients who meet off-treatment criteria, but remain on-study and who are undergoing treatment of toxicity **related to the investigational therapy** (e.g., second transplant for treatment of graft rejection), only adverse events will be recorded and reported for those events which are > grade 3 or serious, are previously unexpected AND are deemed possibly, probably or definitely related to a procedure, test, or treatment, or require intervention. Adverse events resulting from other disease-related treatments not associated with this investigational therapy (i.e. treatment for progression of disease), or from events not related to the patient's disease will NOT be recorded or reported. Expedited reporting will not apply to events arising from participation in another clinical trial or alternative treatment for disease progression.
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6 statistical considerations 6.1 subject

Subjects of both genders, from all racial and ethnic groups are eligible for this trial if they meet the criteria outlined in Section 2.1. To date, there is no information that suggests differences in disease response among racial or ethnic groups or between the genders, indicating that results of the trial will be applicable to all groups. Efforts will be made to extend the accrual to a representative population, but in a pilot study with limited accrual, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic or ineffective treatments on the one hand and the need to explore gender, racial, and ethnic aspects of clinical research on the other. If differences in outcome that correlate to gender, age, racial, or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully.
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6.2 statistics and feasibility

The first primary objective of this study is to assess the feasibility and toxicity associated with infusion of escalating doses of donor derived natural killer (NK) cells (NK-DLI) on Days 21 (± 3 days) and 49 days (± 7 days) following infusion 1, following allogeneic HLA-matched T cell depleted (TCD) peripheral blood stem cell transplantation (PBSCT) in patients with metastatic or recurrent pediatric solid tumors and leukemias. Because of the experience in the first 8 patients transplanted, recipients will now be divided into 2 cohorts: 1) matched related donor recipients and 2) unrelated donor recipients. Patients will be enrolled onto this trial using a conventional Phase I dose escalation design in which 3 patients per dose level, in each cohort are entered, with 3 additional patients enrolled at the dose level, following the usual guidelines for a phase I trial, based on the development of DLTs. After enrollment of the first six patients on study without DLT, cohort 1 was expanded by a maximum of 6 patients to study the pathophysiology of symptoms observed to date. Because no GVHD was seen in related donor recipients (n = 4), dose level 1 for related donor recipients will be closed and patients with related donors will start accrual onto dose level 2. Since the expansion of dose level 1, one additional unrelated donor recipient had been transplanted and developed grade 4 GVHD. Although the GVHD was steroid responsive and therefore did not meet definition of DLT, we find the incidence and severity of the GVHD in these patients unacceptable. Therefore, all subsequent recipients will be treated with GVHD prohylaxis and patients with unrelated donors will continue to enroll on cell dose level 1. In addition to assessment for DLT, if >1 of the additional 3 patients with unrelated donors, enrolled at the cohort 1 cell dose level experience Grade 3 or any experience grade 4 acute GVHD, the NK cell dose level will not be escalated without further consultation with the FDA and IRB regarding a protocol modification. Consistent with a phase I design, the highest dose level at which 0/6 or 1/6 patients has a DLT will be expanded. If at any dose level, there are 2 or more patients who experience a DLT, then no further dose escalations will occur. At the expanded dose level, if 3 or more of 9, or 4 or more of 12 patients experience a DLT, consideration for de-escalation and an expanded cohort at a lower dose level will be given through a protocol amendment. The feasibility objective would be met if >=80% of patients receive 2 doses of NK cell infusions within 56 days of HSCT. As described in the protocol, patients are scheduled to receive NK-DLI on Days 21 (± 3 days) and 49 (± 7 days) but the doses may be delayed based upon clinical judgment of the treating team or concern for toxicity related to previous infusions. Moreover, it is possible that patients may not receive adequate cells because of toxicity, product failure, or product contamination. A second primary objective of this trial is to determine if full donor lymphoid engraftment in T cell depleted, MHC matched sibling or unrelated donor transplants with NK-DLI can be accomplished using this regimen. Donor engraftment is defined as >95% donor lymphoid chimerism (CD3+ T-cells in peripheral blood) on post-BMT day +100. The highest dose explored will enroll 12 total patients in order to allow adequate numbers of patients to receive the regimen for more accurate estimates of engraftment, aGVHD and toxicity at that dose level. Because NK cells are presumed to enhance engraftment rates, only patients enrolled on the highest dose level will be evaluable for the engraftment endpoint. If however, at any dose level, engraftment is problematic and primary graft failures occur in 2 patients, the protocol stopping rule for insufficient engraftment described below will be triggered. It would be desirable to have greater than 75% of patients at the highest NK dose level show sustained donor lymphoid engraftment (>95% at day 100). Thus, if 8 or more of 12 patients engraft, this will be considered adequate since the upper one sided 90% CI about 7/12 is 0.781. A third primary objective of this trial is to determine rates of aGVHD in TCD, MHC matched sibling or unrelated donor transplants given NK-DLI. Upon completion of the trial, the incidence of all aGVHD will be reported with appropriate confidence intervals at all dose levels. All histologies will be analyzed together for this end point, since the incidence of aGVHD is not likely to be influenced by this variable. In addition, however, we will evaluate the incidence of aGVHD as it relates to histology for exploratory purposes in an attempt to identify any unexpected relationships that might exist. A rate of <25% grade III or grade IV acute GVHD per cohort would be required to consider this approach of interest for further development. All patients enrolled, regardless of NK dose level, will be evaluable for the aGVHD endpoint. If however, excessive aGVHD is observed, this will trigger the stopping rules described below.
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6.3 early stopping rules

If the number of patients who have ever been treated on this trial at any dose level 1) develop steroid refractory grade 3 or grade 4 aGVHD at any time point, 2) show primary graft failure - defined as failure to achieve independence from platelet transfusion or ANC never reaching >
500 cells/mcl by day 35 post-BMT, or 3) experience treatment related mortality within 100 days
(defined as toxicity from the preparative regimen, infection, aGVHD or therapy for aGVHD), exceeds 1/5 (upper 90% confidence interval of 58%), 3/10 (upper 90% CI of 55%), or 4/15 (upper 90% CI of 46%) or 6/20 (upper 90% CI of 47%), then accrual to the trial will terminate as soon as this can be evaluated, regardless of how successful the clinical outcomes have been, as this will be considered excessive toxicity.  
In addition, if a single patient (**exception**: patients with bcr-abl+ leukemia) develops a positive bcr-abl PCR confirmed by repeat testing, there will be a temporary suspension to accrual and no products infusions will be given to patients currently on study.
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6.4 subject accrual goal

As stated in section 3.1, three to 6 recipients of unrelated donors will be treated on cell dose level 1, and three to 6 recipients will be treated on cell dose 2 per cohort, with an additional six recipients enrolled on cohort 3, or the highest cohort explored. Thus, a total of up to 38 recipients (and 38 donors) may be enrolled on this trial. Prior to enrolling on the first cohort, a single patient will receive DLI-NK cells once ANC reaches at least 1000 cells/μL or within 42 days following transplant. Due to toxicities observed in the first patient, a second patient received this regimen. Once these first two patients had undergone transplant and accomplished hematopoietic recovery, cohort 1 was opened 28 days after the last NK cell infusion. With a rare possibility of not achieving the targeted cell numbers per cohort, an additional 3 recipient/donor pairs may be enrolled if needed to replace patients who do not meet the targeted cell dose for the assigned cohort, for a maximum of 43 recipients and 43 donors. With an approximately 5-7 patients enrolling per year, anticipated accrual is 3-4 years with 3 year post-transplant follow up. It is therefore likely this trial will remain open for approximately 6 - 8 years.
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7 human subjects protections 7.1

Allogeneic hematopoietic stem cell transplants are being performed at increasing rates each year for patients with malignant diseases. Patients with hematologic malignancies will be the subjects for this study. This patient group was chosen because current treatment options offer little to no hope for survival and new approaches to therapy are desperately needed. While the diseases targeted in this trial affect all races, and genders and subjects of both genders, efforts will be made to extend accrual to a representative population, but in a small pilot trial, it may be difficult if not impossible to achieve complete balance in this regard. The trial will be listed on the NCI and NIH websites and clinical trials search sites. Individuals with HIV disease will not be candidates for this protocol, due to the high risk of post-transplant complications in this population. Individuals who are pregnant or lactating will not be candidates for this protocol, due to risk to the fetus or newborn.
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7.1.2 donors

Related donors will be first-degree HLA-matched family members of the transplant recipient. In most cases the best donor is a sibling. All donors will be selected in accordance with NIH CC Department of Transfusion Medicine standards. Minors will only be selected when they represent the most suitable stem cell donor for the individual recipient. In such cases, the oldest suitable donor will be selected and approval to participate will require medical review by a pediatric practitioner, evaluation by a social worker, and psychological assessment by a mental health specialist with pediatric expertise. For donors who are minors, informed consent from their legal guardian and donor verbal assent will be obtained. Unrelated donors will be HLA-matched (at least 9/10 allele) and will be selected in accordance with National Marrow Donor Program (NMDP) standards.
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7.2 participation of children

The age range of patients eligible for this trial is 4 to 35 years of age. Physicians, nurses, and multidisciplinary support teams have expertise in the management of children undergoing PBSCT. All children enrolled on this protocol will be followed and cared for in the Pediatric Oncology Branch clinic.
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7.3 evaluation of benefits and

Survival for the population of patients eligible for this trial is limited. This population has a very high risk of dying from their disease, and other therapeutic options are primarily experimental. Although the overall treatment plan is experimental, the PBSCT portion of this study is an acceptable therapeutic option for patients in this population, and pre-clinical immunologic studies indicate there may be some benefit to the addition of NK cell administration. The goal of this study is to determine the safety and feasibility of this approach and to determine if transplantation of allogeneic (related or unrelated) HLA-matched NK cells post PBSCT can result in full donor engraftment (>95% by day 100), while reducing the incidence of Graft-versus-Host Disease (GVHD), and reducing the risks associated with infection. Patients participating in this trial will be treated with therapeutic intent, and the response to therapy will be monitored. The potential benefits from PBSCT alone are prolonged disease remission and/or reduction in cancer-related symptoms, while the potential additional benefits of the NK cell infusion are unknown at this time. This protocol involves greater than minimal risk, but does present the potential for direct benefit to individual subjects according to 21CFR50.52, 46.405.
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7.3.1.1 risks associated with pbsct

The primary risks to patients participating in this research study include toxicity of the chemotherapy and immunosuppressive drugs and allogeneic transplantation. The chemotherapy utilized in this study has the risk of direct drug toxicity. In addition, the immune suppression resulting from this therapy is associated with an increased risk of infections. The primary risks associated with allogeneic stem cell transplantation are graft rejection, GVHD and regimen related toxicity. This protocol utilizes a profoundly immunosuppressive induction and preparative regimen designed to facilitate rapid hematologic engraftment. Indeed, patients treated with a similar regimen and T cell depleted graft in the ETIB have shown >90% myeloid engraftment in the majority of patients by day 28 and in all patients by day 98. In addition, the POB and the ETIB have extensive experience with this induction and preparative regimen with T cell replete grafts and have seen universally brisk engraftment, therefore graft rejection is not anticipated to be limiting. In this regimen, melphalan has been added for additional cytoreductive effects. While this may increase the probability that the preparative regimen is myeloablative, it lessens the chance for graft rejection even further and thus we believe that graft rejection is very unlikely in this protocol. Thus, the evidence suggests that GVHD and tumor recurrence/progression will represent the primary cause of morbidity and mortality post-transplant. Because of the history of significant rates of GVHD in previous POB trials, the graft will be T cell depleted and the risk for GVHD is likely to be far lower. The protocol provides for detailed and careful monitoring of all patients to assess for toxicity and response to treatment. All patients entered on the trial will have high risk pediatric malignancy for which standard therapy is ineffective. Further, the chemotherapy drugs used on this protocol are known to be effective for the types of cancer that these patients will have.
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7.3.1.2 risks associated with allogeneic

In previous studies, grade 2 and 3 anemias have been reported though most patients had anemia at study entry. Neutropenia has also been reported, but this was following chemotherapy agents. There were no episodes of neutropenic fever and thrombocytopenia was not observed. Hematologic toxicity was transient and there was no occurrence of prolonged cytopenia or marrow aplasia. Reports suggest that engraftment is improved, rates of GVHD and infections are lower with NK cell infusions, however, a negative impact on engraftment and the development of GVHD are theoretic risks. Based on experiences on this trial, however, there is an apparent increase in GVHD in recipients of unrelated donor products in comparison to recipients of related donor products, which is thought to be at least possibly related to the allogeneic NK cell administration. Because of this disparity, several changes have been made to the protocol to ameliorate the potential risk of NK related GVHD. 1) NK cells are now given no earlier than day 21 (+/- 3) days post PBSCT to avoid concurrent engraftment syndrome which may be exacerbated by NK cell administration; 2) Recipients of unrelated donor products will receive immunosuppression to ameliorate the potential risk of GVHD.
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7.3.1.3 tumor biopsy

In the event a patient has a readily accessible tumor, adult patients may be asked to undergo biopsy in order to obtain tissue for research evaluation (see section **3.2.2**). Standard techniques will be used for percutaneous biopsies and may include CT and / or ultrasound guidance. Although direct benefit from research conducted on this tumor biopsy is unlikely, participation in this research may allow patients some benefit in knowing their contribution may lead to a greater understanding of their cancer and potential benefit to others in the future. Patients enrolled on this protocol suffer from extremely rare malignancies with very low survival rates and often have a strong desire to participate in research that may lead to a better understanding of their disease.
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7.3.2 donors

The donor stem cell collection procedure on this trial is conducted per standard clinical routine. There are a number of potential benefits to donors who participate on this trial. The most probable is psychological benefit from contributing to medical care designed to improve the health of another. Donors appreciate that their participation represents an essential component to a transplant, which is performed with curative intent. This psychological benefit may be derived both at the time of donation, as well as in the future, since it is hoped that the transplant will result in prolongation and/or improvement in the quality of the recipient's life. Donor participation may also help advance scientific knowledge about stem cell transplantation and lead to improvements in the treatment of cancer. (213) The main risks to donors are the possible side effects of stem cell harvesting. Stem cell donation is a safe procedure that is routinely performed in healthy children and adults. The potential side effects of filgrastim used for mobilization are outlined in Section 9.8. The most common side effects of apheresis are pain and bruising at IV sites. Side effects of a temporary central venous catheter, required for young children, include pain, bleeding, bruising, infection, thrombosis, and vascular perforation. Mild side effects from citrate anticoagulant are common and include chills, numbness and tingling sensations ("pins and needles"), anxiety, muscle cramps, and nausea. More serious side effects due to citrate-induced low calcium levels are uncommon and include low blood pressure, seizures, weakness, and tetany. Citrate reactions rapidly resolve away when the collection is slowed down or stopped. Transient mild thrombocytopenia is common after donation. To prevent dilutional anemia, the extracorporeal circuit must be primed with 1 unit of red blood cells for small children. Side effects of blood draws include pain and bruising, lightheadedness, and rarely, fainting. There is the possibility that the donor will have negative psychological effects (e.g., guilt, sadness) if the transplant is unsuccessful or if complications (e.g. GVHD) contribute to recipient morbidity or mortality. Donors will be closely monitored and procedures to minimize risks and prevent side effects are incorporated into all aspects of the protocol. The POB, DTM, and NIH CC and NMDP Donor Sites have broad expertise to adequately manage side effects, including in pediatric donors (related donors only).
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7.4 risk/benefit analysis

All patients enrolled on this study are at very high risk of disease recurrence and from dying of their disease. Patients entered on the trial will be treated with therapeutic intent and response to the therapy and possible toxicity will be closely monitored. Patients on this study may directly benefit from the chemotherapy administered as it may provide anti-tumor activity. Successful allogeneic PBSCT may offer patients a curative therapy for their cancer. It is hypothesized that the addition of NK cell infusions may improve engraftment, reduce the incidence of GVHD and lower infection risks; although these results are not known at this time. Therefore, this protocol involves greater than minimal risk to patients, but presents the potential for direct benefit to individual subjects and therefore meets Federal requirements for patients that are minors. (45CFR46.405)
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7.5 consent processes and documentation

A signed informed consent document will be obtained prior to entry onto the study. The PI or an associate investigator on the trial will obtain consent. The PI, an Associate Investigator, or their designee will be available to answer all questions from patients and their parents or guardians. The investigational nature and research objectives of this trial, the procedures and treatments involved and their attendant risks and discomforts and potential benefits, and alternative therapies will be carefully explained to the patient and/or the patient's parents or guardian if he/she is of minor age. The investigators are requesting a waiver from the IRB to allow only one parent to sign the informed consent to enter a child on the protocol. Because many patients must travel to the NIH from long distances at substantial expense, requiring both parents to be present for the consent process could be a financial hardship for many families. When guardianship status of the child is uncertain, documentation of custody status must be obtained. Where deemed appropriate by the clinician and the child's parent(s) or guardian, the child will also be included in all discussions about the trial and age-appropriate language will be used to describe the procedures and tests involved in this study, along with the risks, discomforts and benefits of participation. Written assent will not be obtained from children as the study holds out the prospect of direct benefit that is important to the health and well-being of the child and is available only in the context of the research. Verbal assent will be obtained as appropriate for children ages > 7 and the parent or guardian will sign the designated line on the informed consent attesting to the fact that the child has given assent. Children under the age of 7 will not be required to provide assent as they typically do not have the cognitive ability to fully understand the nature of research. The consent/assent process will be documented in the child's medical record, including the assessment of the child's ability to provide assent (verbal versus written) as applicable. All children will be contacted after they have reached the age of 18 to determine whether they wish to continue on the trial and informed consent will be obtained from them at that time. 7.5.2 Related donors Informed consent will also be obtained from all donors entered on this trial. Donors < 18 years of age will be evaluated by a social worker and mental health specialist (psychologist or psychiatrist) prior to the assent process to determine willingness to participate. If willingness to participate has been confirmed, verbal assent, as described above, will also be obtained. The attached informed consent documents contain all elements required for consent. In addition, the Principal Investigator, Associate Investigator, or their designee will be available to answer all questions from patients, donors, and their parents or guardians.
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7.5.3 unrelated donors

Informed consent will be obtained from all unrelated donors entered on this trial. This process will be conducted at the NMDP Donor Center using the Protocol Informed Consent Document for Unrelated Donors, which will be approved by both the NCI and NMDP IRBs. Per NMDP regulations, and to maintain donor confidentiality, the signed informed consent document will not be sent to the NIH. Instead, the NMDP will issue a certification notice to the NIH confirming donor consent.
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7.5.4 consent for minors when

When a pediatric subject reaches age 18, continued participation (including ongoing interactions with the subject or continued analysis of identifiable data) will require consenting of the now adult with the standard protocol consent document to ensure legally effective informed consent has been obtained. Given the length of time that has transpired for some of the subjects since their last visit for this study, we request waiver of informed consent for those individuals who have completed their participation in the research study. Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d):
(1) The research involves no more than minimal risk to the subjects.  
a. Analysis of samples and data from this study involves no additional risks to subjects.  
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects.  
a. Retention of these samples or data does not affect the welfare of subjects.  
(3) The research could not practicably be carried out without the waiver or alteration.  
a. Considering the length of time between the minor's last contact with the research team and their age of majority, it will likely be very difficult to locate them again. A significant reduction in the number of samples analyzed is likely to impact the quality of the research.  
(4) Whenever appropriate, the subjects will be provided with additional pertinent information after participation.  
a. We only plan to request a waiver of reconsent for those subjects who have been lost to follow-up or who, prior to the approval of Amendment Q, have been taken off study prior to reaching the age of majority.
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7.5.5 telephone consent

The informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. A witness to the subject's signature will sign and date the consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records. The informed consent process will be documented on a progress note by the consenting investigator and a copy of the informed consent document and note will be kept in the subject's research record.
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8 safety reporting requirements/data and

Any untoward medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject's participation in research, whether or not considered related to the subject's participation in the research.
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8.1.2 suspected adverse reaction

Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 'reasonable possibility' means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug.
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8.1.3 unexpected adverse reaction

An adverse event or suspected adverse reaction is considered "unexpected" if it is not listed in the investigator brochure or is not listed at the specificity or severity that has been observed; or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application. "Unexpected", also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation.
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8.1.4 serious

An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 8.1.5 Serious adverse events An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
x Death, x A life-threatening adverse drug experience x Inpatient hospitalization or prolongation of existing hospitalization  
x A persistent or significant disability/incapacity or substantial disruption of the ability to conduct normal life functions x A congenital anomaly/birth defect. x Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
8.1.6 Disability
A substantial disruption of a person's ability to conduct normal life functions. 8.1.7 Life-threatening adverse drug experience Any adverse event or suspected adverse reaction that places the patient or subject, in the view of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. 8.1.8 Protocol Deviation (NIH Definition) Any change, divergence, or departure from the IRB approved research protocol. 8.1.9 Non-compliance (NIH Definition) The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, IRB requirements, or regulatory requirements for the protection of human research subjects. 8.1.10 Unanticipated Problem Any incident, experience, or outcome that:
x Is unexpected in terms of nature, severity, or frequency in relation to
(a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator's Brochure or other study documents, and
(b) the characteristics of the subject population being studied; AND
x Is related or possibly related to participation in the research; AND  
x Suggests that the research places subjects or others at a *greater risk of harm* (including physical, psychological, economic, or social harm) than was previously known or recognized.
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8.2 nci-irb and clinical director

8.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths The PI will report in the NIH Problem Form to the NCI-IRB and NCI Clinical Director:  
x All deaths, except deaths due to progressive disease x All Protocol Deviations x All Unanticipated Problems
x All non-compliance Reports must be received within 7 days of PI awareness via iRIS. 8.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review The protocol PI will report to the NCI-IRB:  
1. A summary of all protocol deviations in a tabular format to include the date the deviation occurred, a brief description of the deviation and any corrective action.  
2. A summary of any instances of non-compliance 3. A tabular summary of the following adverse events:
a) All Grade 2 **unexpected** events that are possibly, probably or definitely related to the research;
b) All Grade 3 and 4 events that are possibly, probably or definitely related to the research; c) All Grade 5 events regardless of attribution; d) All Serious Events regardless of attribution; NOTE: Grade 1 events are not required to be reported.  
8.2.3 NCI-IRB Reporting of IND Safety Reports Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported to the NCI IRB.
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8.3 ind sponsor reporting criteria

During the first 30 days after the subject receives investigational agent/intervention, the investigator must immediately report to the sponsor, using the mandatory MedWatch form 3500a or equivalent, any serious adverse event, whether or not considered drug related, including those listed in the protocol or investigator brochure and must include an assessment of whether there is a reasonable possibility that the drug caused the event. For serious adverse events that occur more than 30 days after the last administration of investigational agent/intervention, only report those that have an attribution of at least possibly related to the agent/intervention. Required timing for reporting per the above guideline:  
x Deaths (except death due to progressive disease) must be reported via email within 24 hours. A complete report must be submitted within one business day.  
x Other serious adverse events including deaths due to progressive disease must be reported within one business day
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov and to the CCR PI and study coordinator. 8.3.1 Action Plan for Positive Results on Cell Product Safety Testing  
8.3.1.1 In the unlikely event that a positive sterility test or mycoplasma test result is obtained after distribution of the cell product, or after administration of the product to the patient, the following steps will be initiated IMMEDIATELY:
a. CPS, DTM personnel will notify the principal investigator, Dr. Nirali Shah, MD at
(240) 760-6199 or by pager 301-496-1211. As soon as the identification and sensitivity report from positive sterility is available, a copy of the final report will be sent to the PI. The DTM QS officer and CPS Supervisor will determine the need for quality improvement based on the nature and extent of the incident.  
b. If CPS is unable to reach Dr. Shah within 15 minutes, contact the page operator (301496-1211) to page the Pediatric Oncology Branch Fellow on-call. NOTE: Dr. Shah or the POB Fellow on-call will contact the attending physician for the POB Service, who will determine the extent of the work-up of a positive culture in consultation with staff from the Microbiology Service.  
c. Dr. Shah /attending physician will discuss the positive results with the patient/parents, and specify the clinical therapy, antibiotic regimen and/or monitoring plan in consultation with staff from the Microbiology Service.  
d. A contaminated sample of a product that has been administered to a patient will be handled in the same fashion as a Grade 4 toxicity (CTC version 4.0) and the Sponsor will be responsible for notifying the NCI IRB and the FDA. A full written report with description of events, laboratory findings, clinical evaluation and treatments would be submitted to the NCI IRB within 7 working days. The Sponsor will be responsible for filing this report with the FDA within 15 calendar days.
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8.4 data safety monitoring plan

The Principal Investigator will review all adverse events. The Principal Investigator will meet with the research team at frequent and regularly scheduled intervals, to determine treatment modifications and treatment based toxicities, initiating enrollment to the next lower weight based group, to monitor trends in adverse events, and determine if trends are noted. Adverse events will be tracked by the PI. If trends are noted and/or risks warrant it, accrual will be interrupted and/or the protocol and/or consent document will be amended accordingly. If trends in toxicities are noted or stopping rules are met, the PI will temporarily suspend enrollment while reviewing the episodes with the NCI IRB and FDA to determine if any changes to the protocol or the informed consent are warranted. At the completion of each cohort, a toxicity summary with both acute and delayed toxicities will be formally reported to the FDA prior to opening the subsequent cohort. In addition, if a toxicity occurs after D 28 in any patient, that meets DLT criteria, is serious or is unexpected, it will be reported to the FDA immediately, and when applicable, discussions with the FDA and IRB regarding dose adjustments will occur. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff. 8.4.2 Sponsor Monitoring Plan As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring program. The CCR's program allows for confirmation of: study data, specifically data that could affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and SOPs; and human subjects' protection. This is done through independent verification of study data with source documentation focusing on:  
x Informed consent process x Eligibility confirmation x Drug administration and accountability x Adverse events monitoring x Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progress of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct. 8.4.3 Safety Monitoring Committee (SMC) This protocol will require oversight from the Safety Monitoring Committee (SMC). Initial review will occur as soon as possible after the annual NCI-IRB continuing review date. Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting schedule permits or more frequently as may be required by the SMC. For initial and subsequent reviews, protocols will not be reviewed if there is no accrual within the review period. Written outcome letters will be generated in response to the monitoring activities and submitted to the Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI.
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9 pharmaceutical information 9.1 fludarabine

Availability: Fludarabine monophosphate is commercially available and will be supplied by the Clinical Center Pharmacy. FLUDARA IV, is supplied as a white, lyophilized powder. Each vial contains 50 mg of fludarabine phosphate, 50 mg of mannitol, and sodium hydroxide to adjust pH. Fludara is stored at room temperature. Storage and Stability: Reconstituted FLUDARA IV is chemically and physically stable for 24 hours at room temperature, or for 48 hours if refrigerated. Because reconstituted FLUDARA IV contains no antimicrobial preservative, care must be taken to assure the sterility of the prepared solution; for this reason, reconstituted FLUDARA IV should be used or discarded within 8 hours. Preparation: FLUDARA IV should be prepared for parenteral use by aseptically adding Sterile Water for Injection, USP. When reconstituted with 2 ml of Sterile Water for Injection, each ml of the resulting solution will contain 25 mg of Fludarabine Phosphate, 25 mg of mannitol, and sodium hydroxide to adjust the pH to 7-8.5. The product may be further diluted for intravenous administration in 50 ml of 5% Dextrose for Injection USP, or in 0.9% Sodium Chloride, USP. Administration: Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration. Fludarabine will be mixed in 50 ml of 0.9% NaCl, and infused i.v. over 30 minutes. Toxicity: Fludarabine toxicities include myelosuppression (dose limiting toxicity), fever, nausea, vomiting, stomatitis, diarrhea, gastrointestinal bleeding, anorexia, edema, skin rashes, myalgias, headache, agitation, hearing loss, transient episodes of somnolence and fatigue, autoimmune hemolytic anemia, autoimmune thrombocytopenia, paresthesias, peripheral neuropathy, renal, and pulmonary toxicity (interstitial pneumonitis). Severe fatal CNS toxicity presenting with loss of vision and progressive deterioration of mental status has occurred after very high doses (approximately 4 times higher than the standard doses employed in this protocol). Severe neurologic toxicity has only rarely been demonstrated at the 25-30 mg/m2/day dosage of fludarabine. Rarely describe complications include transfusion-associated graft-versus-host disease, thrombotic thrombocytopenic purpura, and liver failure. Tumor lysis syndrome following fludarabine administration has been observed, especially in patients with advanced bulky disease. Opportunistic infections (protozoan, viral, fungal, and bacterial) have been observed post-fludarabine, especially in heavily pre-treated individuals, and in individuals receiving fludarabine combined with other agents.
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9.2 mesna (sodium 2-mercaptoethanesulfonate mesnex)

Availability: Commercially available and will supplied by the Clinical Center Pharmacy. Stability: Mesna may be diluted in D5W, 0.9%NaCl, or D5/0.45NaCl to concentrations between 0.6 mg/ml and 20 mg/ml for intravenous infusions. These concentrations are stable at room temperature for 24 hours. Administration: Will be administered by continuous i.v. infusion on days –6, -5, -4, and –3 of the transplant during high dose cyclophosphamide administration. Dose of mesna will be 1200 mg/m2 per day by continuous i.v. infusion for four consecutive days (days –6, -5, -4, and –3).  
Toxicity May be associated with nausea, vomiting, diarrhea, or headaches.
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9.3 cyclophosphamide (cytoxan)

Availability: Commercially available as a lyophilized powder in 2 gram vials and will supplied by the Clinical Center Pharmacy. Storage and Stability: Following reconstitution as directed, cyclophosphamide is stable for 24 hours at room temperature or 6 days when kept at 2-8C. Preparation: Reconstitution with 100 ml of sterile water for injection to a final concentration of 20 mg/ml. Administration: Cyclophosphamide will be diluted in 0.9% NaCl and infused over 30 minutes during induction and 2-hours during pre-HSCT preparative therapy. Toxicity: Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia and thrombocytopenia. Anorexia, nausea, and vomiting occur, especially after high-dose cyclophosphamide; diarrhea, hemorrhagic colitis, and mucosal and oral ulceration have been reported. Sterile hemorrhagic cystitis occurs in about 20% of patients; severity can range from microscopic hematuria to extensive cystitis with bladder fibrosis. Mesna has been used prophylactically as an uroprotective agent in patients receiving cyclophosphamide. Prophylactic 88 mesna is not effective in preventing hemorrhagic cystitis in all patients. Patients who receive high dose cyclophosphamide may develop interstitial pulmonary fibrosis, which can be fatal. Hyperuricemia due to rapid cellular destruction may occur, particularly in patients with hematologic malignancy. Hyperuricemia may be minimized by adequate hydration, alkalinization of the urine, and/or administration of allopurinol. If allopurinol is administered, patients should be watched closely for cyclophosphamide toxicity (due to allopurinol induction of hepatic microsomal enzymes). At high doses, cyclophosphamide can result in a syndrome of inappropriate antidiuretic hormone secretion; hyponatremia with progressive weight gain without edema occurs. At high doses, cyclophosphamide can result in cardiotoxicity. Deaths have occurred from diffuse hemorrhagic myocardial necrosis and from a syndrome of acute myopericarditis; in such cases, congestive heart failure may occur within a few days of the first dose. Other consequences of cyclophosphamide cardiotoxicity include arrhythmias, potentially irreversible cardiomyopathy, and pericarditis. Other reported adverse effects of cyclophosphamide include headache, dizziness, and myxedema; faintness, facial flushing, and diaphoresis have occurred following IV administration.
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9.4 etoposide (vepesid vp-16)

Availability: Commercially available and will be supplied by the Clinical Center Pharmacy. Etoposide Toxicity: Includes nausea, vomiting, stomatitis, diarrhea, neutropenia, thrombocytopenia, and alopecia. Secondary AML has been associated with etoposide. Bradycardia and hypotension are sometimes observed with etoposide administration.
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9.5 doxorubicin (adriamycin)

Availability: Commercially available and will be supplied by the Clinical Center Pharmacy. Doxorubicin Toxicity: Includes nausea, vomiting, stomatitis, myelosuppression, and cardiotoxicity. Cardiotoxicity is particularly noted after cumulative doses of greater than 550 mg/m2. Other toxicities include nausea, vomiting, stomatitis, diarrhea, and alopecia. Skin infiltration of doxorubicin causes tissue necrosis.
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9.6 vincristine

Availability: Commercially available and will be supplied by the Clinical Center Pharmacy. Vincristine Toxicity Vincristine causes neurological toxicities such as paresthesias, jaw pain, ataxia, foot-drop, cranial nerve palsies, paralytic ileus, constipation, abdominal pain, and loss of deep tendon reflexes. It is also a vesicant, and occasionally causes alopecia and myelosuppression. Because fluconazole can delay the clearance of vincristine, fluconazole will be discontinued during days 1, 2, 3, and 4 of induction chemotherapy. Storage and Stability: Stability studies conducted by the Pharmaceutical Development Service, Pharmacy Department, NIH Clinical Center, have demonstrated that admixtures of vincristine, doxorubicin, and etoposide in 0.9% Sodium Chloride for Injection (at concentrations, respectively, of 1, 25, and 125 mcg/ml; 2, 50, and 250 mcg/ml; and 2.8, 70, and 350 mcg/ml) are stable for at least 36 hours at room temperature when protected from light. Administration of these Agents as Continuous Infusion: In this study, the daily dose of vincristine, doxorubicin, and etoposide (i.e. the 24 hour supply) will be admixed together in 500 ml of 0,9% Sodium Chloride Injection and delivered with a suitable infusion pump through a central venous access device. All 3-in-1 admixtures dispensed from the Pharmacy will contain a 24-hour supply of etoposide, doxorubicin, and vincristine PLUS an overfill (excess) fluid and a proportional amount of drug to compensate for volume lost in administration set tubing to ensure delivery of the entire 24 hour dose. Before dispensing 3-in-1 admixtures, Pharmacy staff will 1) purge all air from the drug product container, 2) attach an administration set appropriate for use with a portable pump and the set will be 3) primed close to its distal tip and 4) capped with a Luer-locking cap. Bags will be exchanged daily for 4 consecutive days to complete a 96-hour drug infusion. Portable pumps used to administer etoposide + doxorubicin + vincristine admixtures will be programmed to deliver 528 ml per day at 22 ml/hr. At the end of an infusion, some residual fluid is expected because overfill fluid and drug were added; however, nurses are asked to return to the Pharmacy for measurement any drug containers that appear to contain a greater amount of residual drug than expected.
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9.7 prednisone

Availability: Commercially available and will be supplied by the Clinical Center Pharmacy; available in solid or liquid dosage forms. Tablet strengths include 1, 2.5, 5, 20, and 50 mg forms, and the oral solution is at 1 mg/ml.  
Administration: Prednisone will be administered at a dose of 60 mg/m2 orally in 2-4 divided doses on days 1, 2, 3, 4, and 5 of fludarabine-EPOCH induction chemotherapy regimen. In patients unable to tolerate oral medication, methylprednisolone can be substituted at the same dosage, diluted in 25 ml of D5W, and infused over 15 minutes. To reduce gastrointestinal side effects, prednisone should be taken with food. Toxicity: Prednisone frequently causes gastritis or ulcers, hypertension, immunosuppression, muscle wasting, fluid retention, increased appetite, psychiatric disturbances, hyperglycemia, and skin changes. In addition, steroids can also cause pancreatitis, impaired growth, menstrual abnormalities, osteoporosis, osteonecrosis, cataracts, pseudotumor cerebri, glaucoma, and seizures.
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9.8 filgrastim (g-csf neupogen)

Availability: Filgrastim is a commercially available and will be supplied by the Clinical Center Pharmacy. This product differs from the natural form due to its absence of N-terminal oglycosylation. Filgrastim has undergone tests for USP pyrogen, limulus amebocyte assay, sterility and the general safety test. The nucleic acid content can be no greater than 10 pg/unit dose. The final product is clear, colorless sterile solution that is free of particulates; the product is not less than 95% pure. It is provided in 1 ml vials of 300 mcg or 1.6 ml vials of 480 mcg. Storage and Stability: Intact vials should be stored in the refrigerator; do not freeze. Product is stable for at least 1 year when refrigerated. Preparation: Do not dilute with sodium chloride solutions. Administration: Inject subcutaneously at 5-10 mcg/kg/day as per treatment plan. Toxicity: Filgrastim may cause bone pain, constitutional symptoms such as fever, fatigue and myalgias, splenomegaly, thinning hair, pain at the injection site, bone pain, worsening of preexisting inflammatory conditions, and elevations in uric acid, LDH, and alkaline phosphatase.
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9.9 pegfilgrastim (recombinant methionyl human

Availability: Pegfilgrastim is a commercially available and will be supplied by the Clinical Center Pharmacy. Pegfilgrastim is a covalent conjugate of recombinant methionyl human GCSF (filgrastim) and monomethoxypolyethylene glycol. To produce pegfilgrastim, a 20 kD
monomethoxypolyethylene glycol molecule is covalently bound to the N-terminal methionyl residue of filgrastim. The average molecular weight of pegfilgrastim is approximately 39 kD. Pegfilgrastim is supplied in 0.6 mL prefilled syringes for subcutaneous injection, containing 6 mg pegfilgrastim (based on protein weight), in a sterile, clear, colorless, preservative-free solution (pH 4.0) containing acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), and sodium (0.02 mg) in water for injection, USP. Storage and Stability: Pegfilgrastim should be stored refrigerated and protected from light until time of use. Shaking or freezing should be avoided. Before injection, Pegfilgrastim may be allowed to reach room temperature for a maximum of 48 hours but should be protected from light. Administration: Inject subcutaneously as per treatment plan. Toxicity: See filgrastim above.
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9.10 cytarabine (cytosine arabinoside cytosar

Availability: Commercially available. Storage and Stability: The reconstituted drug is stable for 48 hours at room temperature. The drug will be further diluted in an age appropriate amount of 5% Dextrose Injection or 0.9% Sodium Chloride Injection prior to administration. Administration: Cytarabine is available for intravenous and intrathecal administration. The manufacturer supplies the drug as unreconstituted lyophilized powder in vials. With each vial, an ampule of bacteriostatic water for injection containing 0.945% benzyl alcohol is provided. To prepare high dose (2 gm/m2) cytarabine intravenous infusions, each vial of drug should be reconstituted with 2 cc (100 mg) or 10 cc (500 mg) of sterile water for injection USP (i.e. not the manufacturer-provided diluent) which contains no preservative. Begin each cytarabine dose 3.5 hours after completion of the preceding fludarabine dose. Toxicity: IV Administration: Acute DLT consists of severe leukopenia and thrombocytopenia. Nausea and vomiting may be dose limiting at higher doses. Other adverse reactions include diarrhea, immunosuppression, anorexia, stomatitis, oral ulceration, flu-like syndrome, fever, hepatic dysfunction, and alopecia. At high doses, as in this protocol, keratoconjunctivitis, dermatitis, and central nervous system toxicity (e.g., ataxia, somnolence, coma, dysarthria) may occur. Occasionally, the CNS impairment is not fully reversible. Renal impairment will enhance toxicity. In the event signs of CNS toxicity, the cytarabine will be interrupted and the M.D. notified. In the event of signs of CNS toxicity, the cytarabine infusion will be interrupted and the M.D. notified. No further cytarabine will be administered if there is CNS toxicity (any grade) deemed related to cytarabine. Conjunctivitis Prophylaxis: Corticosteroid ophthalmic drops will be administered 2 drops to each eye every 6 hours starting prior to first dose and continuing until 24 hours after the last dose of cytarabine has been completed.
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9.11 intrathecal chemotherapy 9.11.1 methotrexate

Source and Pharmacology: A folate analogue that inhibits the enzyme dihydrofolate reductase, halting DNA, RNA, and protein synthesis. Initial IV half-life is about 1.2 hours, with a second phase of 10.4 hours. About 50% is bound to protein. Transport into the cell is carrier-mediated. Once in the cell, MTX (Glu)n are formed, the number of which are related to the cytocidal effect. Once MTX (Glu)n are formed, they do not pass back out of the cell unless converted back to MTX. After oral administration, about 60% of a 30 mg/m2 dose is rapidly absorbed from the GI tract, with peak blood levels at 1 hour. Above this dose, absorption decreases significantly. Absorption can be very erratic, varying between 23% to 95%. A 20-fold difference between peak levels of drug has been reported (0.1 to 2mM). There is significant enterohepatic circulation of MTX: 9% of MTX is excreted in feces. MTX is excreted unchanged in the urine, except at high doses when it is partially metabolized to hydroxy-MTX and excreted. Formulation and Stability: Intact vials may be stored at room temperature (22°-25°C) and are stable for at least 2 years or until date of expiration. INTRATHECAL MTX: Available in various dosages in preservative-free liquid, 25mg/ml in a 2ml vial, or as a lyophilized powder. Reconstitute the powder with buffered saline solution. The Methotrexate solutions may be further diluted with buffered saline or the patient's own CSF. After mixing it should be used within 24 hours, since MTX contains no antibacterial preservative. Guidelines for Administration: The drug will be filtered and mixed in sterile preservative free saline less than 4 hours prior to administration. Intrathecal delivery should be approximately isovolumetric (ml CSF out = ml drug in) via lumbar puncture (LP) in the lateral decubitus position. The patient should remain in prone or Trendelburg position for approximately 30 minutes post LP to facilitate drug circulation throughout the CNS. Leucovorin rescue should be administered 5 mg PO/IV q 6 hours x 4 doses, starting 24 hours after methotrexate. Supplier: Commercially available. See package insert for further information. Toxicities: The following toxicities may occur when methotrexate is given alone into the spinal fluid:  
| Common                                       | Occasional                                                                                    | Rare                                                                                                                                |
|----------------------------------------------|-----------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------|
| Happens to 21-100  children out of every 100 | Happens to 5-20 children  out of every 100                                                    | Happens to < 5 children  out of every 100                                                                                           |
| Immediate:                                   | Headache, abnormally high  number of cells in the spinal  fluid                               | Vomiting, fever, rash,  drowsiness, stiff neck,  irritation of tissues in the  brain/spinal cord,  seizures (L), partial  paralysis |
| Within 1-2 days of receiving drug Prompt:    | Decrease in the number  of red and white blood  cells and platelets made  in the bone marrow, |                                                                                                                                     |  
92  
| Version Date: 07/23/2018 Within 2-3 weeks,  prior to the next  course   | drowsiness, unsteady  walk                                                                                                                                   |
|-------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Learning disability                                                     | Thinning of nerve fiber  insulation resulting in  difficulty with loss of  memory, concentration,  balance and walking,  which may get worse  with time1 (L) |
| Delayed: Any time later  during therapy Late:                           | Increasingly poor  nervous system function1                                                                                                                  |
| Any time after the  completion of  treatment                            |                                                                                                                                                              |
| 1 May be enhanced by HDMTX and/or cranial irradiation.                  |                                                                                                                                                              |  
(L) Toxicity may also occur later.  
Methotrexate crosses the placenta to the fetus. Fetal toxicities and teratogenic effects of methotrexate (either alone or in combination with other antineoplastic agents) have been noted in humans. The toxicities include: congenital defects, chromosome abnormalities, malformation, severe newborn myelosuppression, pancytopenia, and low birth weight.  
Dose Modification: Hold for any CNS toxicity.  
9.11.2 Hydrocortisone (Cortef, Solu-cortef) Route: Intrathecal therapy (as clinically indicated) Source and Pharmacology: Commercially available. See package insert for further information. Synthetic steroid akin to natural adrenal hormone, cortisol. It binds with steroid receptors on nuclear membrane, impairs cellular mitosis and inhibits protein synthesis. It is phase specific, killing cells primarily during S phase. It has a catabolic effect on proteins and alters the kinetics of peripheral blood leukocytes. It is excreted in the urine and catabolized in the liver. Toxicity: The following toxicities may occur when hydrocortisone is given intrathecally: nausea and vomiting, headache, pleocytosis, fever, somnolence, meningismus, learning disability, leukoencephalopathy. Formulation and Stability: Available as 100 mg, 250 mg, 500 mg, and 1000 mg vials for aqueous injection. In powder form, the drug is stable for 2 years at room temperature. After reconstitution, it is stored at room temperature, and should be discarded after 3 days. Intrathecal Administration: IT hydrocortisone should be reconstituted with physiologic buffered diluents (lactated Ringer's, 0.9% sodium chloride, Elliott's B solution). Do not use Bacteriostatic Water to reconstitute for IT use; use only preservative free solutions.  
Guidelines for Administration: Hydrocortisone will be given by IT administration in an agespecified dose and will be mixed with cytarabine.  
9.11.3 Cytarabine (cytosine arabinoside, AraC, Cytosar) Route: Intrathecal therapy (as clinically indicated) Source and Pharmacology: Commercially available. See package insert for further information. Deoxycytidine analogue which is metabolized to ARA-CTP, a substance which inhibits DNA
polymerase. It is S phase specific, and thus affects DNA synthesis.  
Toxicity: The following toxicities may occur when cytarabine is given intrathecally: Nausea, vomiting, headache, pleocytosis, arachnoiditis, rash, fever, somnolence, meningismus, convulsions, paresis, myelosuppression, ataxia, learning disability. CNS impairment may not be fully reversible. No further cytarabine will be administered if there is CNS toxicity (any grade) deemed related to cytarabine. Formulation and Stability: A freeze-dried powder available in 100 mg, 500 mg, 1 G and 2 G vials. The unreconstituted form of the drug is stable at room termperature for at least 2 years. Intrathecal Administration: IT cytarabine should be reconstituted with physiologic buffered diluents (lactated Ringer's, 0.9% sodium chloride, Elliott's B solution). Do not use Bacteriostatic Water to reconstitute for IT use; use only preservative free solutions. Guidelines for Administration: Cytarabine will be given by IT administration in an age-specified dose and will be mixed with hydrocortisone.
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9.12 ranitidine (zantac)

Will be given as a pre-medication. It will be supplied by the Clinical Center Pharmacy. The most common side effects are fatigue, dizziness, headache, and diarrhea. See package insert for additional information.
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9.13 acetaminophen (tylenol)

Will be given as a pre-medication. It will be supplied by the Clinical Center Pharmacy. The most common side effect seen with high doses or chronic usage is hepatotoxicity. Rarely, sensitivity reactions can occur. See package insert for additional information.
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9.14 diphenhydramine (benadryl)

Will be given as a pre-medication IV over 10-15 minutes. It will be supplied by the Clinical Center Pharmacy. The most common side effects are sleepiness, dizziness, restlessness, and irritability. See package insert for additional information.
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9.15 fluconazole (diflucan)

Will be given as prophylaxis against susceptible yeast and fungi. It will be supplied by the Clinical Center Pharmacy. Fluconazole increases the AUC and serum concentration of tacrolimus and sirolimus. Thus, careful monitoring of levels is important when these medications are administered concurrently. Significant interactions with oral hypoglycemics, coumarin-type anticoagulants, phenytoin, rifampin, theophylline, cisapride, astemizole, rifabutin and tacrolimus have been observed. As described in section **3.3.2.7**, interactions with vincristine also occur. Rare mild side effects including headache, nausea and abdominal pain have been reported. Elevations in transaminases can occur. Hepatotoxicity is usually, but not always, reversible. Fluconazole has been associated with rare cases of severe hepatic toxicity, including fatalities. Other adverse effects include anaphylaxis, and rash (occasionally severe). See package insert for additional information.
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9.16 pcp prophylaxis

9.16.1 Trimethoprim/Sulfamethoxazole (TMP/SMX) (Bactrim, Septra): Will be given as prophylaxis against pneumocystis. It will be provided by the Clinical Center Pharmacy. Side effects include a reduction in blood counts, gastrointestinal disturbances (nausea, vomiting and anorexia) and allergic reactions. Interactions with thiazide diuretics, phenytoin, and methotrexate occur and require caution when used concurrently. Due to photosensitivity, patients should avoid prolonged exposure to the sun. Fatalities associated with the administration of sulfonamides, although rare, have occurred due to Stevens-Johnson syndrome, toxic epidermal necrolysis, fulminant hepatic necrosis and blood dyscrasias. See package insert for additional information.
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9.16.2 pentamadine

Will be given as prophylaxis against pneumocystis, as a substitute for TMP/SMX. It will be provided by the Clinical Center Pharmacy. It is supplied in 300 mg per nebulizer. Dosing will be as per the CC BMT Guidelines (http://intranet.cc.nih.gov/bmt/clinicalcare/pdf/Table_VII.pdf). Side effects reported with the use of Pentamidine include metallic taste, coughing, bronchospasm in heavy smokers and asthmatics; increased incidence of spontaneous pneumothorax in patients with previous PCP infection or pneumatoceles, or hypoglycemia. IV formulation may be used if there is a contraindication to inhaled delivery.
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9.17 intravenous immunoglobulin (ivig gamimmune

Will be used for infection prophylaxis in the post-transplant period. It will be provided by the Clinical Center Pharmacy and will be administered by standard guidelines. Side effects vary depending on the preparation but can included anaphylactic reactions, hypotension, fever, chills, headache, urticaria, and aseptic meningitis. In general, these effects are infusion-related and can be treated by decreasing the rate of infusion. Rarely, acute renal failure has been reported, predominantly in individuals with pre-existing renal dysfunction. Although donor screening and inactivation procedures substantially reduce the risk of viral transmission, immune globulin is made from human plasma and, therefore, carries a risk of transmission of infectious agents. See package insert for the individual preparations for additional information.
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9.18 palifermin kepivance™️ (kgf)

Kepivance™ (palifermin) is a human keratinocyte growth factor (KGF) produced by recombinant DNA technology in *Escherichia coli (E coli).* It will be supplied by the Clinical Center Pharmacy. See the package insert for complete details. Palifermin is supplied as a sterile, white, preservative-free, lyophilized power for IV injection after reconstitution with 1.2 mL of sterile water for injection, USP. Palifermin will be administered to decrease the incidence and duration of oral mucositis. It may be administered at 60 mcg/kg/day IV bolus injection for 3 days, twice:
1. The first 3 daily doses should be administered prior to the preparative chemotherapy, with the 3rd dose at least 24 hours prior to starting the preparative regimen, 2. The last 3 daily doses should be administered starting on day -1.  
Toxicities associated with use of palifermin include: rash, erythema, edema, pruritus, dysesthesia, tongue discoloration, tongue thickening, alteration of taste, pain arthralgias and dysesthesia.
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9.19 recombinant human interleukin 15

rhIL-15 will be purchased by the POB Branch from CellGenix™ for use in cell preparation. rhIL-15 is produced under GMP conditions for investigational use; the source is a DNA
sequence encoding the mature IL-15 protein expressed in *E. coli.* For clinical *ex vivo* cell culture use only.
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formulation and stability

Lyophilized from a 250 mcg/mL, 0.2 mcm-sterile filtered solution in PBS (300 mM NaCl) + 0.1% Tween 20. Each vial contains 50 mcg (+/- 10%). Product is stable for 2 years from date of production when stored as directed. This study will require approximately 2 vials per patient for cell development. Storage: Store vials at -20°C to -80°C; avoid repeated freeze-thaw cycles.
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9.20 cyclosporine a

Availability: Cyclosporine A is commercially available as either an injectable concentrate or as a microemulsion in capsules or liquid. Capsules are available in 25 and 100 mg strengths. Liquid solution is available as 100 mg/ml. Storage and Stability: Stable at room temperature. Administration: Cyclosporine will be started by IV infusion at day –1 before the transplant at a dose of 2 mg/kg/dose (over a two hour period) for two doses per day. Once the patient is able to tolerate oral feedings, CSA will be converted to an equivalent oral dose. The starting oral dose should be approximately 2-times the IV dose given in a q 12 hour schedule. The dose should be titrated to a trough level of 150-300 ng/ml (monoclonal RIA methodology). Drug Interactions Cyclosporine has important interactions with multiple medications. Most commonly, CSA levels are altered due to induction or inhibition of hepatic metabolism. Azole antifungal agents (e.g., fluconazole), macrolides (e.g., erthyromycin), ciprofloxacin, metronidazole, grapefruit juice, cimetidine, metoclopramide, oral contraceptive pills, and corticosteroids may result in elevated cyclosporin A levels. Agents that decrease CSA levels include nafcillin, anticonvulsants (e.g., phenytoins, barbituates), and octreotide. CSA levels should be monitored more closely when any such agents are co-administered, in the setting of impaired renal or hepatic function, and when other nephrotoxic or hepatoxic drugs are used. Cisapride is contraindicated due to risk of QT prolongation and arrhythmias. Toxicity: Includes nephrotoxic effects, which are manifested as increases in BUN and creatinine, hypertension, hyperkalemia, and hyperuricemia; nephrotoxicity is increased by co-administration of acyclovir, aminoglycoside antibiotics, and amphotericin B. Nervous system effects include a fine hand tremor, seizures, headache, paresthesis, flushing, and confusion. Cyclosporine-induced seizures may be associated with concurrent high-dose corticosteroid therapy, hypertension, and hypomagnesemia. Rarely, cyclosporine can induce a syndrome of severe neurotoxicity characterized by cortical blindness, quadriplegia, seizures, and coma. Adverse dermatologic effects include hirsutism and gingival hyperplasia. Elevated liver transaminases may occur. Adverse GI effects include diarrhea, nausea and vomiting, anorexia and abdominal discomfort.
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9.21 tacrolimus (fk506 prograf)

Supply: Tacrolimus will be obtained by the NIH Clinical Center Pharmacy Department from commercial sources and is available in capsules (0.5 mg, 1mg, and 5mg), and as a parenteral concentrate for injection (5 mg/ml, 1 ml ampules). Preparation: For parenteral doses, tacrolimus injection concentrate (5 mg/ml) should be diluted to a final concentration of 0.004 to 0.02 mg/ml in dextrose 5% in water or sodium chloride 0.9%.  
Parenteral doses of tacrolimus will be prepared in non-PVC containers and infused with nonPVC administration sets/tubing.  
Storage and Stability: Capsules and ampules of parenteral concentrate bear expiration dates and are stored at room temperature. Tacrolimus concentrate for injection that has been diluted to a final concentration of 0.004 to 0.02 mg/ml is stable for 24 hours in 5% dextrose or 0.9% sodium chloride injection in glass, PVC or non-PVC plastic containers. To minimize the potential for sorption to PVC plastic bags and tubing as well the leaching of phthalate plasticizer (DEHP) into the solution, only non-PVC plastic bags and intravenous administration sets should be utilized. Administration: Tacrolimus may be given intravenously over 24 hours or orally. Toxicities: Acute tacrolimus nephrotoxicity is usually manifested by a moderate decline in renal excretory function, which is readily reversible by a decrease in drug dosage. Although some degree of transient renal dysfunction may occur in patients with therapeutic levels of tacrolimus, significant renal toxicity is associated with elevated trough or steady state levels. In addition to an increase in BUN and creatinine, hyperkalemic hyperchloremic acidosis, low fractional excretion of sodium and the onset of hypertension with hypomagnesemia are seen with tacrolimus nephrotoxicity. Insulin dependent post transplant diabetes mellitus was reported in 20% kidney transplant patients, which was reversible in 50% of the patients at 2 years post transplant. Hypertension occurs in up to 60% of patients. Hypomagnesaemia can be associated with neurological symptoms, including seizures, cerebellar ataxia and depression. Dose-related hepatotoxicity, manifested by elevation of serum transaminases and bilirubin, has been reported. The drug carries a warning for increased susceptibility to infection and the possible development of lymphoma may result from resulting immunosuppression.
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9.22 busulfan (busulfex)

General: Busulfex is a bifunctional alkylating agent approved for use as a conditioning agent prior to allogeneic hematopoieitc stem cell transplantation. The IV formulation is administered as Busulfex (Busulfex, Otsuka America Phamraceutical, Inc.) Supply: For patient administration, IV Busulfex is purchased by the NIH Clinical Center Pharmacy Department from commercial sources. The drug is supplied as a clear, colorless sterile solution in 10ml single use vials. Each vial of BUSULFEX contains 60 mg (6mg/ml) of busulfan. Storage and Stability: Unopened vials of BUSUILFEX are stable until the date indicated on the package when stored under refrigeration at 2-6 degrees C (36-46 degrees F). 97 Administration: BUSULFEX must be diluted prior to use with either 0.9% Sodium chloride or 5% Dextrose Injection. The diluent quantity should be 10 times the volume of BUSULFEX so that the final concentration of busulfan is approximately 0.5 mg/ml. BUSULFEX should be administered intravenously via a central venous catheter as a two hour infusion every 24 hours for a total of 4 doses over four days. Toxicities: 1) At the indicated dose and schedule BUSULFEX induces profound myelosuppression; 2) All patients should be pre-medicated with phenytoin or Clonazepam plus Levetiracetam (Keppra) since busulfan is known to cross the blood brain barrier and induce seizures; 3) Nausea, vomiting, and stomatitis are common side-effects of busulfan. Drug Interactions: Itraconazole increases the clearance of busulfan by up to 25%. Phenytoin increases the clearance of busulfan by 15%.
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9.23 levetiracetam (keppra)

Supply: 500 mg tablets Preparation: None Storage and Stability: Tablets are stored at room temperature. Route of Administration: Orally. Children: the dose is 10 mg/kg/dose PO BID (maximum dose:
1000 mg). Adults: drug will be given as 500mg po every 12 hours. Dosing starts the night before BUSULFEX and continues until 24 hours after last Busulfan dose. Toxicities: Leukopenia, hepatic abnormalities.
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9.24 lorezapam (ativan)

General: benzodiazepine Administration: Oral or IV, 0.02-0.05 mg/kg/dose, maximum dose: 2 mg) given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours after last busulfan dose. Toxicity: CNS depression, confusion, paroxysimal agitation.
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9.25 radiation therapy 9.25.1 total

Chemical name: Not applicable Known toxicities: The side effects of radiation have been well described. The most common include nausea and mucositis. There also exists a risk of hypothyroidism, cataracts, interstitial pneumonitis, nephropathy, and an unspecified long-term risk of developing secondary malignancies. Importantly, the majority of the non-neoplastic effects were subclinical and/or reversible. Studies attempting to evaluate the risk attributed to radiation within a preparative regimen have implicated secondary malignancies, however this is difficult to ascribe since secondary malignancies are also increased with cGVHD and other post-transplant complications. Pediatric patients who require sedation in order to undergo TBI will be asked to sign a separate consent outlining the risk of the selected anesthesia. 9.25.2 CNS Boost
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chemical name not applicable

Known toxicities: For the CNS boost, the common short-term side effects include skin redness and irritation in the area treated, hair loss in the area treated, fatigue, and uncommonly headaches, weakness, numbness, or other neurologic deficit which may require steroid treatment, nausea, vomiting, weight loss, and rarely decrease blood cell count and excessive need of sleep. Long-term side effects of whole-brain radiation therapy, which may occur months to years after, are mild decrease in memory or ability to think (which may be exacerbated in those who have received prior radiation therapy to the brain), scalp discoloration and thickening, permanent thinning of the hair in the treated area. Uncommonly, severe memory loss, cataracts, permanent loss of brain cells, seizures, hearing loss. Rarely, death of brain cells can cause swelling in the brain and require long-term steroid treatment, hospitalization and/or surgery, loss or decrease in vision. Extremely rare is development of new tumors in the scalp, skull or brain.