Clinical trial NCT01848457
Conditions: Osteosarcoma Nephrotoxicity Ototoxicity
Keywords: Osteosarcoma Cancer Nephrotoxicity Ototoxicity Cisplatin High-dose methotrexate Biomarkers Patient reported outcomes
Sponsor: Children's Hospital of Philadelphia
Start date: 2013-04-01; Completion date: 2016-10-01
Enrollment: 13 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Osteosarcoma is the most common type of bone cancer in children, adolescents and young adults. Treatment with surgery and a combination of three conventional chemotherapy drugs can cure nearly two-thirds patients with osteosarcoma, but the treatment can also cause irreversible damage to the kidneys and cause permanent hearing loss. The purpose of this study is to evaluate new approaches to prevent these side effects without interfering with the beneficial effects of the chemotherapy drugs on the cancer by using our knowledge of how the drugs damage the kidney and cochlear hair cells in the ear to selectively block these side effects. Preventing these side effects without interfering with the anti-cancer effect of the drugs will improve the outcome in survivors and may also improve the effectiveness of the chemotherapy regimen by preventing treatment delays and dose reductions that are often caused by the side effects. Patients will be carefully monitored to ensure that the new interventions do not adversely affect response to the treatment and do not increase the other side effects of the chemotherapy. Specifically, we will monitor the nutritional status of the patients closely and ask patients to complete a survey describing the side effects after each treatment cycle. We will also collect a small sample of cancer tissue at the time of biopsy and surgery from each patient on this study for testing to determine new classes of anti-cancer drugs currently under development may have a role in treating osteosarcoma. If effective, these new approaches to prevent kidney damage and hearing loss will be applicable in other types of cancers treated with the same chemotherapy drugs.

Detailed description:
Current osteosarcoma treatment regimens include cisplatin and high-dose methotrexate (HDMTX), which are nephrotoxic and ototoxic, and the damage to kidneys and cochlear hair cells may be irreversible. Preventing these toxicities will improve the outcome in long-term survivors and may also prevent short-term treatment delays and dose reductions that can compromise the efficacy of the treatment regimen and allow for administration of higher cumulative doses of cisplatin. This pilot study evaluates pharmacologically-based approaches to prevent the nephrotoxic effect of HDMTX by prolonging the infusion duration and thereby lowering the risk of drug precipitation in renal tubules; and to selectively block the uptake of cisplatin into renal tubular cells and cochlear hair cells by inhibiting the organic cation transporter 2 (OCT2) with the proton pump inhibitor (PPI), pantoprazole. Participants with previously untreated biopsy-proven, localized or metastatic osteosarcoma will receive six cycles of the standard Methotrexate, Adriamycin (doxorubicin),cisplatin (MAP) chemotherapy regimen, which includes high-dose methotrexate, doxorubicin and cisplatin. The first 2 cycles are administered neoadjuvantly followed by surgery to remove the primary tumor, when feasible.

A novel randomized, crossover, 2 x 2 factorial clinical trial design allows all patients to receive the new interventions to prevent toxicity and to serve as their own controls. New, sensitive urinary biomarkers of acute kidney injury serve as primary endpoints for evaluating treatment-related renal damage. Ototoxicity will be monitored using audiograms. The effect of these interventions on tumor response (radiographic and histologic) and toxicity (including a patient reported outcome survey and nutritional status) will be closely monitored. Other secondary objectives include evaluating bone-specific alkaline phosphatase as a biomarker of tumor burden and constructing a tissue microarray to evaluate expression of proteins that are responsible for resistance to the current drugs used to treat osteosarcoma and assess expression of proteins that are targeted by new anticancer drugs under development for childhood cancers.

Arms: arm 1: Cycles 1 and 2: HDMTX administered as a 4-hour infusion and pantoprazole is administered with cisplatin Cycles 3 and 4: HDMTX administered as a 12-hour infusion and cisplatin is administered alone arm 2: Cycles 1 and 2: HDMTX administered as a 4-hour infusion and cisplatin is administered alone Cycles 3 and 4: HDMTX administered as a 12-hour infusion and pantoprazole is administered with cisplatin arm 3: Cycles 1 and 2: HDMTX administered as a 12-hour infusion and pantoprazole is administered with cisplatin Cycles 3 and 4: HDMTX administered as a 4-hour infusion and cisplatin is administered alone arm 4: Cycles 1 and 2: HDMTX administered as a 12-hour infusion and cisplatin is administered alone Cycles 3 and 4: HDMTX administered as a 4-hour infusion and pantoprazole is administered with cisplatin
Interventions: intervention 1: Pantoprazole intervention 2: High-dose methotrexate infusion duration
Intervention descriptions: intervention 1: 0.3 mg/kg IV over 15 min immediately prior to cisplatin as a loading dose on days 1 \& 2 followed by 1.3 mg/kg IV infused over 4 h concurrent with the 4 h cisplatin infusion on days 1 \& 2 of treatment Cycles 1 \& 2 (Treatment Arms 1, 3) OR Cycles 3 \& 4 (Treatment Arms 2, 4) intervention 2: High-dose methotrexate (12 g/sq m, maximum dose 20 g) will be infused over 4 hours or 12 hours

Protocol documents:
Prot_SAP_000.pdf: 
	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
i	  
	  
Title:	   Pilot	   Study	   to	   Prevent	   the	   Nephrotoxicity	   of	   High-­‐Dose	   Methotrexate	   by	  
Prolonging	   the	   Infusion	   Duration	   and	   Prevent	   the	   Nephrotoxicity	   and	  
Ototoxicity	   of	   Cisplatin	   with	   Pantoprazole	   in	   Children,	   Adolescents	   and	  
Young	  Adults	  with	  Osteosarcoma	  	  
Study	  Key	  Name:	   CHP12ST051	  OS	  Nephrotoxicity	  and	  Ototoxicity	  
Protocol	  Number:	   CHP12ST051	  
Drug	  Name:	   Pantoprazole	  
FDA	  IND:	   Exempt	  
Protocol	  Date:	   April	  2,	  2013	  
Amendments:	   Amendment	  1	  date:	  
Amendment	  2	  date:	  
Sponsor:	   The	  Gateway	  for	  Cancer	  Research	  
Principal	  
Investigator:	  
Frank	  M.	  Balis,	  M.D.	  
Center	  for	  Childhood	  Cancer	  Research	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB	  4024	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐426-­‐5414	  
FAX:	  267-­‐425-­‐0113	  
Email:	  balisf@email.chop.edu	  
Co-­‐Investigators:	   Rochelle	  Bagatell,	  M.D.	  
Center	  for	  Childhood	  Cancer	  Research	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB	  4022	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐426-­‐9737	  
Email:	  bagatellr@email.chop.edu	  
	   Naomi	  Balamuth,	  M.D.	  
Center	  for	  Childhood	  Cancer	  Research	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB	  10050	  
Philadelphia,	  PA	  19104	  
Phone:	  215-­‐590-­‐3437	  
Email:	  balamuth@email.chop.edu	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
ii	  
	   John	  P.	  Dormans,	  M.D.	  
Chief,	  Division	  of	  Orthopedics	  
Department	  of	  Surgery	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3401	  Civic	  Center	  Blvd	  
Philadelphia,	  PA	  19104	  
Phone:	  215-­‐590-­‐1527	  
Email:	  dormans@email.chop.edu	  
	   Elizabeth	  Fox,	  M.D.	  
Center	  for	  Childhood	  Cancer	  Research	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB	  4022	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐425-­‐3010	  
Email:	  foxe@email.chop.edu	  
	   Bruce	  Pawel,	  M.D.	  
Department	  of	  Pathology	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
34th	  and	  Civic	  Center	  Blvd.	  
Philadelphia,	  PA	  19104	  
Phone:	  215-­‐590-­‐1762	  
Email:	  pawelb@email.chop.edu	  
	   Lisa	  States,	  M.D.	  
Department	  of	  Radiology	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3401	  Civic	  Center	  Blvd.	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐425-­‐7146	  
Email:	  states@email.chop.edu	  
Richard	  Womer,	  M.D.	  
Center	  for	  Childhood	  Cancer	  Research	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB	  10409	  
Philadelphia,	  PA	  19104	  
Phone:	  215-­‐590-­‐2229	  
Email:	  womer@email.chop.edu	  
	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
iii  
Statistician:	  
Study	  Coordinator:	  
Yimei	  Li,	  Ph.D.	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB10207	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐425-­‐3084	  
Email:	  liy3@email.chop.edu	  
Sarah	  Strange	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB104021	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐425-­‐3142	  
Email:	  stranges@email.chop.edu	  
Research	  Nurse:	  
	  
Theresa	  DiSipio	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
3501	  Civic	  Center	  Blvd.	  CTRB4023	  
Philadelphia,	  PA	  19104	  
Phone:	  267-­‐426-­‐8194	  
Email:	  disipiot@email.chop.edu	  
	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
iv  
	  TABLE	  OF	  CONTENTS	  
ABBREVIATIONS	  AND	  DEFINITIONS	  OF	  TERMS	  ............................................................................	  vii	  
PROTOCOL	  SYNOPSIS	  ....................................................................................................................	  ix	  
SCHEDULE	  OF	  STUDY	  PROCEDURES	  ............................................................................................	  xiii	  
STUDY	  DIAGRAM	  ..........................................................................................................................	  xv	  
1.	   BACKGROUND	  INFORMATION	  AND	  RATIONALE	  .....................................................................	  1	  
1.1	   Introduction	  .......................................................................................................................	  1	  
1.2	   Compliance	  Statement	  ......................................................................................................	  1	  
1.3	   Relevant	  Literature	  and	  Data	  ............................................................................................	  2	  
1.3.1	   Osteosarcoma	  .............................................................................................................	  2	  
1.3.2	   Nephrotoxicity	  ............................................................................................................	  3	  
1.3.3	   Biomarkers	  of	  Acute	  Kidney	  Injury	  .............................................................................	  7	  
1.3.4	   Ototoxicity	  ..................................................................................................................	  9	  
1.3.5	   Pantoprazole	  ..............................................................................................................	  9	  
1.3.6	   Serum	  biomarkers	  for	  osteosarcoma	  .......................................................................	  11	  
1.3.7	   Study	  Rationale	  .........................................................................................................	  11	  
2.	   STUDY	  OBJECTIVES	  ................................................................................................................	  13	  
3.	   INVESTIGATIONAL	  PLAN	  ........................................................................................................	  13	  
3.1	   General	  Schema	  of	  Study	  Design	  .....................................................................................	  13	  
3.1.1	   Screening,	  Diagnosis,	  and	  Staging	  ............................................................................	  14	  
3.1.2	   Baseline	  (Pretreatment)	  Research	  Studies	  ...............................................................	  15	  
3.1.3	   Treatment	  with	  MAP	  Regimen	  .................................................................................	  15	  
3.1.4	   Criteria	  for	  Starting	  Treatment	  .................................................................................	  18	  
3.1.5	   Dose	  Modifications	  ...................................................................................................	  19	  
3.1.6	   Local	  Control	  of	  Primary	  Tumor	  ...............................................................................	  19	  
3.1.7	   Management	  of	  Metastatic	  Tumors	  .........................................................................	  20	  
3.2	   Randomization	  .................................................................................................................	  20	  
3.3	   Study	  Duration,	  Enrollment	  and	  Number	  of	  Sites	  ...........................................................	  20	  
3.3.1	   Duration	  of	  Study	  .....................................................................................................	  20	  
3.3.2	   Number	  of	  Subjects	  and	  Number	  of	  Study	  Sites	  ......................................................	  20	  
3.4	   Study	  Population	  .............................................................................................................	  21	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
v  
3.4.1	   Study	  Enrollment/Registration	  .................................................................................	  21	  
3.4.2	   Inclusion	  Criteria	  .......................................................................................................	  21	  
3.4.3	   Exclusion	  Criteria	  ......................................................................................................	  22	  
4.	   RESEARCH	  PROCEDURES	  .......................................................................................................	  22	  
4.1	   Research	  Studies	  during	  Treatment	  ................................................................................	  22	  
4.2	   Post-­‐Therapy	  Follow-­‐up	  ..................................................................................................	  23	  
4.3	   Concomitant	  Medications	  ...............................................................................................	  23	  
4.4	   Subject	  Completion/Withdrawal	  .....................................................................................	  23	  
5.	   STUDY	  ENDPOINTS	  AND	  EVALUATIONS	  ................................................................................	  24	  
5.1	   Primary	  Endpoints	  ...........................................................................................................	  24	  
5.2	   Secondary	  Endpoints	  .......................................................................................................	  24	  
5.3	   Efficacy	  Evaluations	  .........................................................................................................	  25	  
5.4	   Safety	  Evaluations	  ...........................................................................................................	  25	  
6.	   STATISTICAL	  CONSIDERATIONS	  .............................................................................................	  26	  
6.1	   Statistical	  Analysis	  Plan	  ...................................................................................................	  26	  
6.1.1	   Primary	  Endpoints	  ....................................................................................................	  26	  
6.1.2	   Secondary	  Endpoints	  ................................................................................................	  27	  
6.2	   Sample	  Size	  and	  Power	  ....................................................................................................	  28	  
6.3	   Interim	  Analysis	  ...............................................................................................................	  29	  
7.	   STUDY	  MEDICATIONS	  ............................................................................................................	  30	  
7.1	   Intravenous	  Pantoprazole	  (PROTONIX	  I.V.)	  .....................................................................	  30	  
8.	   SAFETY	  MANAGEMENT	  .........................................................................................................	  31	  
8.1	   Clinical	  adverse	  events	  ....................................................................................................	  32	  
8.2	   Adverse	  Event	  Reporting	  .................................................................................................	  32	  
8.3	   Definition	  of	  an	  Adverse	  Event	  ........................................................................................	  32	  
8.4	   Definition	  of	  a	  Serious	  Adverse	  Event	  (SAE)	  ....................................................................	  32	  
8.4.1	   Relationship	  of	  SAE	  to	  study	  drug	  or	  other	  intervention	  or	  procedure	  ...................	  33	  
8.4.2	   IRB/IEC	  Notification	  of	  SAEs	  .....................................................................................	  33	  
8.4.3	   Follow-­‐up	  report	  .......................................................................................................	  33	  
9.	   STUDY	  ADMINISTRATION	  ......................................................................................................	  33	  
9.1	   Data	  collection	  and	  management	  ...................................................................................	  33	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
vi  
9.1.1	   Confidentiality	  ..........................................................................................................	  33	  
9.1.2	   Security	  .....................................................................................................................	  34	  
9.1.3	   Anonymization,	  de-­‐identification,	  or	  destruction	  ....................................................	  34	  
9.2	   Confidentiality	  .................................................................................................................	  34	  
9.3	   Regulatory	  and	  ethical	  considerations	  ............................................................................	  34	  
9.3.1	   Data	  safety	  and	  monitoring	  plan	  ..............................................................................	  34	  
9.3.2	   Risk	  assessment	  ........................................................................................................	  34	  
9.3.3	   Potential	  benefits	  of	  trial	  participation	  ....................................................................	  35	  
9.3.4	   Risk-­‐benefit	  assessment	  ...........................................................................................	  35	  
9.4	   Recruitment	  strategy	  .......................................................................................................	  35	  
9.5	   Informed	  consent/assent	  ................................................................................................	  35	  
9.6	   Payments	  to	  subjects/families	  ........................................................................................	  36	  
10.	   	  PUBLICATION	  ......................................................................................................................	  36	  
11.	   REFERENCES	  ........................................................................................................................	  36	  
APPENDIX	  1	   SAMPLE	  TREATMENT	  DELIVERY	  MAP	  ....................................................................	  43	  
APPENDIX	  2	   PEDIATRIC	  NEUROTOXICITY	  GRADING	  SCALE	  .......................................................	  44	  
APPENDIX	  3	   PATIENT	  REPORTED	  OUTCOME	  SURVEY	  ...............................................................	  46	  
	  
	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
vii	  
ABBREVIATIONS	  AND	  DEFINITIONS	  OF	  TERMS	  
	  
Term	  
Definition	  
AKI	  
Acute	  kidney	  injury	  
AUC	  
Area	  under	  the	  plasma	  concentration-­‐time	  curve	  
Block	  
Two	  consecutive	  treatment	  cycles	  (Cycles	  1	  &	  2	  or	  Cycles	  3	  &	  4)	  
BMP	  
Basic	  mineral	  panel	  
BSAP	  
Bone-­‐specific	  alkaline	  phosphatase	  
CBC	  
Complete	  blood	  counts	  
CL	  
Clearance	  
Cmax	  
Peak	  concentration	  
CMP	  
Comprehensive	  mineral	  panel	  
COG	  
Children’s	  Oncology	  Group	  
Course	  
A	  course	  of	  cisplatin	  is	  60	  mg/m2	  daily	  x	  2	  days	  (total	  dose	  120	  mg/m2);	  a	  course	  
of	  doxorubicin	  is	  37	  mg/m2	  daily	  x	  2	  days	  (total	  dose	  75	  mg/m2);	  a	  course	  of	  
HDMTX	  is	  two	  12	  g/m2	  infusions	  administered	  1	  week	  apart	  
Cr	  
Creatinine	  
CrCL	  
Creatinine	  clearance	  
CR	  
Complete	  Response	  
Cycle	  
A	  course	  of	  cisplatin	  and	  doxorubicin	  followed	  3	  weeks	  later	  by	  a	  course	  of	  
HDMTX	  (cycles	  1-­‐4;	  duration	  5	  weeks);	  OR	  A	  course	  of	  doxorubicin	  followed	  2	  
weeks	  later	  by	  a	  course	  of	  HDMTX	  (cycles	  5&6;	  duration	  4	  weeks)	  	  
EOI	  
End	  of	  infusion	  
EOT	  
End	  of	  therapy	  
FEMg	  
Fraction	  excretion	  of	  magnesium	  
GFR	  
Glomerular	  filtration	  rate	  
H&P	  
History	  and	  physical	  exam	  
HDMTX	  
High-­‐dose	  methotrexate	  (12	  g/m2)	  
IV	  
Intravenous	  
KIM-­‐1	  
Kidney	  Injury	  Molecule-­‐1	  	  
LV	  
Leucovorin	  
MAP	  
Methotrexate,	  Adriamycin	  (doxorubicin),	  cisPlatin	  
Mg	  
Magnesium	  
MTX	  
Methotrexate	  
NAG	  
N-­‐Acetyl-­‐β-­‐D-­‐Glucosamidase	  
NGAL	  
Neutrophil	  Gelatinase-­‐Associated	  Lipocalin	  
OCT2	  
Organic	  cation	  transporter	  2	  
OS	  
Osteosarcoma	  
PK	  
Pharmacokinetics	  
Pre-­‐op	  
Preoperatively	  
PPI	  
Proton	  pump	  inhibitor	  
PR	  
Partial	  Response	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
viii	  
Pre-­‐Rx	  
Pretreatment	  
PRO	  
Patient	  reported	  outcome	  
PS	  
Performance	  score	  (Lansky	  or	  Karnofsky)	  
T1/2	  
Half-­‐life	  
TDM	  
Treatment	  delivery	  map	  (roadmap)	  
UA	  
Urinalysis	  
	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
ix	  
PROTOCOL	  SYNOPSIS	  
STUDY	  TITLE	  
Pilot	  Study	  to	  Prevent	  the	  Nephrotoxicity	  of	  High-­‐Dose	  Methotrexate	  by	  Prolonging	  
the	  Infusion	  Duration	  and	  Prevent	  the	  Nephrotoxicity	  and	  Ototoxicity	  of	  Cisplatin	  with	  
Pantoprazole	  in	  Children,	  Adolescents	  and	  Young	  Adults	  with	  Osteosarcoma	  
SPONSOR	  
The	  Gateway	  for	  Cancer	  Research	  
CLINICAL	  PHASE	  
Pilot	  clinical	  trial	  
PARTICIPATING	  SITES	  
The	  Children’s	  Hospital	  of	  Philadelphia	  
STUDY	  RATIONALE	  
Current	   osteosarcoma	   treatment	   regimens	   include	   drugs	   that	   are	   nephrotoxic	   and	  
ototoxic,	   and	   the	   damage	   to	   kidneys	   and	   cochlear	   hair	   cells	   may	   be	   irreversible.	  
Preventing	  these	  toxicities	  will	  improve	  the	  outcome	  in	  long-­‐term	  survivors	  and	  may	  
also	  prevent	  short-­‐term	  treatment	  delays	  and	  dose	  reductions	  that	  can	  compromise	  
the	   efficacy	   of	   the	   treatment	   regimen	   and	   allow	   for	   administration	   of	   higher	  
cumulative	   doses	   of	   cisplatin.	   This	   pilot	   study	   evaluates	   pharmacologically-­‐based	  
approaches	  to:	  
• prevent	   the	   nephrotoxic	   effect	   of	   high-­‐dose	   methotrexate	   (HDMTX)	   by	  
prolonging	   the	   infusion	   duration	   and	   thereby	   lowering	   the	   risk	   of	   drug	  
precipitation	  in	  renal	  tubules,	  and	  
• selectively	  block	  the	  uptake	  of	  cisplatin	  into	  renal	  tubular	  cells	  and	  cochlear	  hair	  
cells	  by	  inhibiting	  the	  organic	  cation	  transporter	  2	  (OCT2)	  with	  the	  proton	  pump	  
inhibitor,	  pantoprazole.	  
These	  pharmacologically-­‐based	  approaches	  to	  prevent	  nephrotoxicity	  and	  ototoxicity	  
may	  also	  be	  applicable	  in	  other	  cancers	  treated	  with	  cisplatin	  or	  HDTX.	  	  
STUDY	  OBJECTIVES	  
Primary	  objective	  
The	   primary	   objective	   is	   to	   identify	   more	   rational,	   pharmacologically-­‐based	   drug	  
delivery	   approaches	   to	   prevent	   nephrotoxicity	   from	   HDMTX	   and	   cisplatin	   and	  
ototoxicity	  from	  cisplatin	  in	  patients	  with	  OS	  by:	  
• comparing	   biomarkers	   of	   acute	   kidney	   injury	   (AKI),	   glomerular	   function	   and	  
plasma	  and	  urine	  MTX	  concentrations	  after	  12	  g/m2	  of	  MTX	  infused	  over	  4	  and	  
12	  hours,	  
• comparing	   biomarkers	   of	   AKI,	   glomerular	   function	   and	   fractional	   excretion	   of	  
magnesium	   (FEMg)	   after	   cisplatin	   administered	   with	   and	   without	   the	   OCT2	  
inhibitor,	  pantoprazole,	  and	  
• comparing	   audiograms	   after	   cisplatin	   administered	   with	   and	   without	  
pantoprazole.	  
Secondary	  objectives	  
• compare	  the	  incidence	  and	  severity	  (grade)	  of	  common	  toxicities	  from	  cisplatin/	  
doxorubicin	   administered	   with	   and	   without	   pantoprazole	   and	   from	   HDMTX	  
administered	  as	  a	  4	  and	  12	  h	  infusion,	  
• compare	   radiographic	   response	   (log	   ratio	   of	   tumor	   volumes)	   and	   histological	  
response	   (percent	   tumor	   necrosis)	   to	   two	   cycles	   (10	   weeks)	   of	   neoadjuvant	  
chemotherapy	  for	  the	  two	  infusion	  durations	  of	  HDMTX	  and	  for	  cisplatin	  with	  
and	  without	  pantoprazole,	  
• correlate	  urinary	  markers	  of	  AKI	  and	  GFR	  estimated	  from	  serum	  cystatin	  C	  to	  
standard	   measures	   of	   renal	   function	   (serum	   creatinine,	   urinalysis,	   estimated	  
creatinine	  clearance,	  FEMg),	  
• build	  a	  tissue	  microarray	  from	  biopsy,	  primary	  resection	  and	  resected	  metastatic	  
tumors	  to	  evaluate	  the	  expression	  of	  proteins	  that	  are	  responsible	  for	  resistance	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
x	  
to	  the	  drugs	  in	  the	  MAP	  regimen	  and	  to	  assess	  expression	  of	  proteins	  that	  are	  
targeted	  by	  new	  anticancer	  drugs	  under	  development	  for	  childhood	  cancers,	  
• evaluate	  serum	  BSAP	  as	  a	  potential	  biomarker	  for	  OS,	  	  
• monitor	  nutritional	  status	  (weight,	  arm	  circumference,	  skin	  fold	  thickness,	  pre-­‐
albumin)	  in	  patients	  receiving	  the	  MAP	  chemotherapy	  regimen,	  and	  
• pilot	  an	  osteosarcoma-­‐specific	  patient	  reported	  outcomes	  survey	  to	  assess	  the	  
incidence	  and	  severity	  of	  tumor-­‐related	  and	  treatment-­‐related	  symptoms.	  
TEST	  ARTICLE	  
Pantoprazole	  
STUDY	  DESIGN	  
A	  randomized	  cross-­‐over,	  2	  x	  2	  factorial	  design,	  in	  which	  patients	  serve	  as	  their	  own	  
control,	  will	  be	  used	  to	  maximize	  the	  potential	  to	  detect	  differences	  in	  biomarkers	  of	  
AKI	   (NAG,	   NGAL,	   KIM-­‐1,	   cystatin	   C)	   and	   audiograms	   resulting	   from	   altering	   the	  
infusion	  duration	  of	  HDMTX	  and	  from	  adding	  pantoprazole	  to	  cisplatin.	  
Two	   infusion	   durations	   of	   HDMTX	   will	   be	   studied	   –	   the	   standard	   4	   h	   duration	   and	  
more	  prolonged	  12	  h	  infusion	  duration.	  Leucovorin	  rescue	  will	  commence	  24	  h	  from	  
the	  start	  of	  the	  infusion.	  The	  HDMTX	  infusions	  will	  be	  divided	  into	  2	  blocks	  (cycles	  1	  &	  
2,	   cycles	   3	   &	   4),	   each	   of	   which	   include	   4	   HDMTX	   infusions.	   The	   order	   of	   infusion	  
duration	  studied	  in	  each	  block	  will	  be	  randomly	  determined.	  
Pantoprazole	  will	  either	  be	  administered	  with	  the	  first	  two	  courses	  of	  cisplatin	  during	  
cycles	  1	  &	  2	  or	  with	  the	  third	  and	  fourth	  courses	  during	  cycles	  3	  &	  4,	  and	  the	  order	  
will	   be	   assigned	   by	   randomization.	   Cisplatin	   is	   administered	   as	   a	   60	   mg/m2	   dose	  
infused	  over	  4	  h	  daily	  x	  2	  d.	  At	  steady	  state,	  7.2	  mg/m2	  of	  active	  cisplatin	  is	  in	  the	  
body,	  and	  by	  the	  end	  of	  the	  4	  h	  infusion,	  88%	  of	  the	  total	  dose	  has	  been	  cleared.	  
Therefore,	  adequate	  concentrations	  to	  inhibit	  OCT2	  should	  be	  present	  for	  at	  least	  4	  h	  
to	  prevent	  cisplatin	  nephrotoxicity	  and	  ototoxicity.	  Pantoprazole	  has	  a	  short	  half-­‐life	  
(1.2	  h),	  and	  when	  administered	  at	  a	  dose	  of	  1.6	  mg/kg	  as	  a	  standard	  15	  min	  infusion,	  
plasma	   pantoprazole	   concentration	   by	   the	   end	   of	   the	   4	   h	   cisplatin	   infusion	   is	   1.3	  
mcM.	  By	  administering	  the	  same	  dose	  as	  a	  0.3	  mg/kg	  loading	  dose	  infused	  over	  15	  
min	   followed	   by	   a	   4	   h	   infusion	   of	   the	   remaining	   1.3	   mg/kg,	   plasma	   pantoprazole	  
concentration	  is	  5.9	  mcM	  at	  4	  h.	  
The	   primary	   endpoint	   to	   assess	   nephrotoxicity	   will	   be	   the	   AKI	   markers	   (cystatin	   C,	  
KIM-­‐1,	  NGAL,	  NAG)	  and	  fractional	  excretion	  of	  magnesium	  (FEMg).	  This	  will	  allow	  us	  
to	  assess	  each	  course	  of	  HDMTX	  and	  cisplatin	  independently.	  Standard	  renal	  function	  
tests	  (urinalysis,	  serum	  creatinine,	  estimated	  creatinine	  clearance,	  and	  FEMg)	  will	  also	  
be	  monitored.	  Audiograms	  will	  be	  performed	  after	  each	  course	  of	  cisplatin	  to	  assess	  
ototoxicity.	  Radiographic	  and	  histological	  response	  in	  the	  resected	  tumor	  specimen	  
after	  neoadjuvant	  chemotherapy	  will	  be	  assessed	  to	  ensure	  that	  changes	  in	  HDMTX	  
infusion	   duration	   and	   the	   addition	   of	   pantoprazole	   to	   cisplatin	   do	   not	   alter	   the	  
efficacy	  of	  chemotherapy.	  
A	  brief	  survey	  to	  assess	  the	  patient’s	  view	  of	  disease-­‐related	  and	  drug	  toxicity-­‐related	  
symptoms	  will	  be	  piloted	  prior	  to	  each	  treatment	  cycle,	  post-­‐op	  and	  at	  the	  end	  of	  
therapy,	  and	  nutritional	  status	  will	  be	  monitored	  over	  the	  course	  of	  therapy.	  
The	  use	  of:	  
• sensitive,	   short-­‐term	   biomarkers	   as	   endpoints	   to	   assess	   the	   effect	   of	  
interventions, 
• continuous,	   rather	   than	   categorical,	   variables	   that	   are	   analyzed	   by	   course	   or	  
cycle	  rather	  than	  by	  patient,	  and 
• a	  crossover	  design	  with	  patients	  serving	  as	  their	  own	  control,	  
will	  minimize	  the	  number	  of	  subjects	  and	  time	  required	  to	  complete	  this	  pilot	  study	  in	  
a	  limited	  patient	  population	  with	  a	  rare	  form	  of	  cancer,	  increase	  statistical	  power,	  and	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
xi	  
maximize	  the	  amount	  of	  data	  collected	  in	  this	  pilot	  study.	  
SUBJECT	  POPULATION	  
Children,	  adolescents	  and	  young	  adults	  with	  previously	  untreated	  osteosarcoma	  
Inclusion	  Criteria	  
• Age:	  <30	  years	  of	  age	  
• Diagnosis:	  histological	  diagnosis	  of	  high-­‐grade	  osteosarcoma	  
• Site:	   Extremity	   or	   central	   axis	   (including	   craniofacial)	   primary	   tumor;	   localized	   or	  
metastatic	  
• Prior	   therapy:	   No	   prior	   chemotherapy	   or	   radiation	   therapy	   for	   osteosarcoma.	  
Subjects	  who	  develop	  osteosarcoma	  as	  a	  second	  cancer	  are	  eligible	  if	  they	  have	  not	  
previously	  received	  cisplatin,	  doxorubicin	  or	  MTX	  
• Kidney	  function:	  Normal	  serum	  creatinine	  for	  age	  and	  gender	  
• Cardiac	  function:	  Shortening	  fraction	  on	  echocardiogram	  >28%	  
• Hearing:	   Hearing	   level	   threshold	   ≤25	   dB	   at	   all	   frequencies	   in	   both	   ears	   to	   be	  
evaluable	  for	  assessment	  of	  pantoprazole’s	  effect	  on	  cisplatin	  ototoxicity.	  Patients	  
with	  hearing	  loss	  can	  be	  enrolled	  but	  will	  not	  be	  evaluable	  for	  ototoxicity	  objective.	  
• Hematological	  function:	  Absolute	  neutrophil	  count	  >1,000/mcL	  and	  platelet	  count	  
>100,000/mcL	  
Exclusion	  Criteria	  
• Receiving	   H2	   antagonists	   (cimetidine,	   ranitidine,	   famotidine,	   nizatidine)	   or	   proton	  
pump	  
inhibitors	  
(lansoprazole,	  
omeprazole,	  
pantoprazole,	  
esomeprazole,	  
raberprazole,	  dexlansoprazole)	  AND	  unable	  to	  hold	  the	  drug	  for	  24	  h	  prior	  to	  and	  24	  
h	  after	  each	  cisplatin	  course	  on	  cycles	  1-­‐4.	  
• Pregnant	  or	  breastfeeding	  
• Unable	  to	  cooperate	  with	  research	  procedures	  
NUMBER	  OF	  SUBJECTS	  
24	  
DURATION	  OF	  SUBJECT	  
PARTICIPATION	  
40	  weeks	  
STUDY	  DURATION	  
24-­‐36	  months	  
STUDY	  PHASES	  
Pilot	  clinical	  trial	  
EFFICACY	  EVALUATION	  
Radiographic	  and	  histological	  response	  to	  neoadjuvant	  therapy,	  survival	  
PHARMACOKINETIC	  
EVALUATION	  
Plasma	  and	  urine	  methotrexate	  concentrations	  at	  the	  end	  of	  each	  HDMTX	  infusion	  on	  
cycles	  1-­‐4	  
SAFETY	  EVALUATION	  
Nadir	   neutrophil	   count	   and	   duration	   of	   severe	   (ANC	   <500/mcL)	   neutropenia,	   nadir	  
platelet	   count	   and	   number	   of	   platelet	   transfusions,	   hospital	   admissions/days	   for	  
neutropenia	  with	  fever	  or	  infection,	  highest	  value	  for	  serum	  ALT	  and	  total	  bilirubin,	  
grade	  3	  or	  4	  mucositis,	  treatment	  delays	  or	  missed	  chemotherapy	  doses	  to	  allow	  for	  
recovery	   from	   toxicity,	   dose	   modifications	   for	   toxicity,	   and	   deaths	   attributed	   to	  
chemotherapy	  toxicity	  will	  be	  monitored	  and	  collected	  by	  cycle.	  
After	  the	  first	  12	  patients	  have	  completed	  therapy,	  the	  incidence	  and	  severity	  of	  the	  
events	   listed	   above	   as	   well	   as	   the	   nephrotoxicity	   and	   ototoxicity	   endpoints	   will	   be	  
assessed	  by	  Cycle	  according	  to	  HDMTX	  infusion	  duration	  (4	  h	  vs.	  12	  h)	  and	  according	  
to	  whether	  or	  not	  IV	  pantoprazole	  was	  administered	  with	  cisplatin	  to	  ensure	  that	  the	  
experimental	  dosing	  methods	  are	  not	  associated	  with	  excessive	  toxicity.	  
After	   the	   first	   12	   patients	   have	   undergone	   resection	   of	   their	   primary	   tumor,	   the	  
radiographic	  response	  (log	  ratio)	  and	  histologic	  response	  (%	  necrosis)	  will	  be	  assessed	  
according	  to	  HDMTX	  infusion	  duration	  (4	  h	  vs.	  12	  h)	  during	  Cycles	  1	  &	  2	  and	  according	  
to	  whether	  or	  not	  IV	  pantoprazole	  was	  administered	  with	  cisplatin	  during	  Cycles	  1	  &	  2	  
to	   ensure	   that	   the	   anti-­‐tumor	   effect	   of	   the	   MAP	   chemotherapy	   regimen	   is	   not	  
compromised	  by	  the	  experimental	  dosing	  methods.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
xii  
STATISTICAL	  AND	  
ANALYTIC	  PLAN	  
This	  pilot	  study	  uses	  a	  2	  x	  2	  factorial,	  randomized	  crossover	  design,	  which	  will	  allow	  
patients	  to	  serve	  as	  their	  own	  control.	  Nephrotoxicity	  data	  will	  be	  analyzed	  by	  course	  
for	  cisplatin	  and	  by	  dose	  for	  HDMTX,	  and	  ototoxicity	  data	  will	  be	  analyzed	  by	  cycle.	  	  
The	   primary	   endpoints	   are	   sensitive	   urinary	   biomarkers	   of	   AKI,	   standard	   renal	  
function	   tests,	   and	   audiograms,	   and	   all	   endpoints	   will	   be	   analyzed	   as	   continuous	  
variables.	  
Twenty-­‐four	  patients	  will	  be	  enrolled	  and	  patients	  serve	  as	  their	  own	  control.	  Over	  
the	   past	   5	   years,	   57	   newly	   diagnosed	   patients	   with	   osteosarcoma	   were	   seen	   and	  
treated	  at	  CHOP.	  This	  is	  an	  average	  of	  11.5	  patients	  per	  year,	  indicating	  that	  we	  can	  
complete	   accrual	   to	   this	   study	   in	   about	   two	   years.	   For	   each	   primary	   endpoint,	  
separate	  linear	  mixed	  effects	  models	  will	  be	  constructed	  for	  comparison	  of	  cisplatin	  
with	  vs.	  without	  pantoprazole	  and	  for	  comparison	  of	  the	  4	  vs.	  12	  h	  infusion	  durations	  
of	  HDMTX.	  
DATA	  AND	  SAFETY	  
MONITORING	  PLAN	  
The	  research	  team	  led	  by	  the	  protocol	  PI	  will	  monitor	  adverse	  events	  related	  to	  the	  
protocol	   therapy	   in	   real	   time.	   The	   progress	   of	   every	   patient	   actively	   receiving	  
treatment	  on	  this	  study	  will	  be	  discussed	  weekly	  in	  the	  Solid	  Tumor	  Team	  Meeting.	  
An	  interim	  analysis	  to	  assess	  the	  safety	  of	  the	  interventions	  will	  be	  performed	  after	  
the	  first	  12	  patients	  have	  completed	  therapy.	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
xiii  
SCHEDULE	  OF	  STUDY	  PROCEDURES	  
PROCEDURE	  
Baseline	  
Cycle	  1	  
Cycle	  2	  
Surgery	  
Cycle	  3	  
Cycle	  4	  
Cycle	  5	  
Cycle	  6	  
End	  therapy	  
Standard	  of	  care	  procedures	  
Tumor	  biopsy	  
X	  
	  
	  
	  
	  
	  
	  
	  
	  
H&P,	  PS,	  weight,	  height	  
X	  
D1,	  22,	  29	  
D1,	  22,	  29	  
Pre-­‐op	  
D1,	  22,	  29	  
D1,	  22,	  29	  
D1,	  15,	  22	  
D1,	  15,	  22	  
X	  
CBC	  +	  Differential	  
X	  
D1	  &	  weekly	  
D1	  &	  weekly	  
Pre-­‐op	  
D1	  &	  weekly	  
D1	  &	  weekly	  
D1	  &	  weekly	  
D1	  &	  weekly	  
X	  
CMP	  +	  PO4,	  Mg	  
X	  
D1,	  22	  
D1,	  22	  
Pre-­‐op	  
D1,	  22	  
D1,	  22	  
D1,	  15	  
D1,	  15	  
X	  
BMP	  
	  
D29	  
D29	  
	  
D29	  
D29	  
D22	  
D22	  
	  
UA	  w/o	  micro	  
X	  
D1,	  221,	  291	  
D1,	  221,	  291	  
	  
D1,	  221,	  291	  
D1,	  221,	  291	  
D1,	  151,	  221	  
D1,	  151,	  221	  
X	  
Urine	  pregnancy	  test2	  
X	  
	  
	  
	  
	  
	  
	  
	  
	  
Serum	  creatinine3	  
	  
D22,	  29	  
D22,	  29	  
	  
D22,	  29	  
D22,	  29	  
D15,	  22	  
D15,	  22	  
	  
Serum	  MTX3	  
	  
D22,	  29	  
D22,	  29	  
	  
D22,	  29	  
D22,	  29	  
D15,	  22	  
D15,	  22	  
	  
Creatinine	  clearance4	  
X	  
	  
	  
	  
Pre-­‐Rx	  
	  
Pre-­‐Rx	  
	  
X	  
Echocardiogram5	  
X	  
	  
	  
	  
Pre-­‐Rx	  
	  
Pre-­‐Rx	  
	  
X	  
Audiogram	  
X	  
	  
Pre-­‐Rx	  
	  
Pre-­‐Rx	  
Pre-­‐Rx	  
	  
	  
X	  
Plain	  X-­‐ray	  of	  primary	  
X	  
	  
	  
Pre-­‐op	  
Pre-­‐Rx	  
	  
	  
	  
X	  
MRI	  of	  primary	  
X	  
	  
	  
Pre-­‐op	  
Pre-­‐Rx6	  
	  
	  
	  
X6	  
Chest	  CT	  scan	  
X	  
	  
	  
Pre-­‐op	  
	  
	  
Pre-­‐Rx7	  
	  
X	  
Bone	  or	  PET	  scan	  
X	  
	  
	  
Pre-­‐op	  
	  
	  
Pre-­‐Rx7	  
	  
X7	  
Dexrazoxane	  
	  
D1,	  2	  
D1,	  2	  
	  
D1,	  2	  
D1,	  2	  
D1,	  2	  
D1,	  2	  
	  
Doxorubicin	  
	  
D1,	  2	  
D1,	  2	  
	  
D1,	  2	  
D1,	  2	  
D1,	  2	  
D1,	  2	  
	  
Cisplatin	  
	  
D1,	  2	  
D1,	  2	  
	  
D1,	  2	  
D1,	  2	  
	  
	  
	  
Pegfilgrastim	  
	  
D3	  
D3	  
	  
D3	  
D3	  
D3	  
D3	  
	  
HDMTX	  +	  LV	  rescue	  
	  
D22,	  29	  
D22,	  29	  
	  
D22,	  29	  
D22,	  29	  
D15,	  22	  
D15,	  22	  
	  
Tumor	  resection	  
	  
	  
	  
X	  
	  
	  
	  
	  
	  
%	  Tumor	  necrosis	  
	  
	  
	  
X	  
	  
	  
	  
	  
	  
Research	  procedures	  
IV	  Pantoprazole	  
	  
D1,	  28	  
D1,	  28	  
	  
D1,	  28	  
D1,	  28	  
	  
	  
	  
Urinary	  AKI	  markers,	  Cr9	  
X	  
D1,	  2,	  7,	  22,	  
23,	  29,	  30	  
D1,	  2,	  7,	  22,	  
23,	  29,	  30	  
Pre-­‐op	  
D1,	  2,	  7,	  22,	  
23,	  29,	  30	  
D1,	  2,	  7,	  22,	  
23,	  29,	  30	  
D1	  
	  
	  
Serum	  Cystatin	  C10	  
X	  
D1,	  7,	  22,	  29	  
D1,	  7,	  22,	  29	  
Pre-­‐op	  
D1,	  7,	  22,	  29	  
D1,	  7,	  22,	  29	  
D1	  
	  
X	  
Plasma/Urinary	  Mg,	  Cr11	  
X	  
D1,	  2,	  7	  
D1,	  2,	  7	  
	  
D1,	  2,	  7	  
D1,	  2,	  7	  
D1	  
	  
X	  
BSAP	  
X	  
	  
	  
Pre-­‐op	  
D1	  
	  
	  
	  
X	  
Pre-­‐albumin	  
X	  
	  
D1	  
Pre-­‐op	  
D1	  
D1	  
D1	  
D1	  
X	  
Urine	  MTX12	  
	  
EOI	  D22,	  29	  
EOI	  D22,	  29	  
	  
EOI	  D22,	  29	  
EOI	  D22,	  29	  
	  
	  
	  
PRO	  survey13	  
X	  
	  
D1	  
Pre-­‐op	  
D1	  
D1	  
D1	  
D1	  
X	  
Arm	  circum/Skin	  fold	  
X	  
	  
D1	  
Pre-­‐op	  
D1	  
D1	  
D1	  
D1	  
X	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
xiv	  
1	  
Check	  urine	  pH	  prior	  to	  the	  start	  of	  HDMTX	  and	  every	  shift	  (q8h)	  until	  serum	  MTX	  <1	  mcM.	  
2	  
For	  females	  ≥11	  years	  of	  age	  
3	  
At	  the	  end	  of	  the	  HDMTX	  infusion,	  hour	  24,	  and	  daily	  until	  MTX	  <0.1	  mcM	  
4	  
12	  or	  24	  hour	  urine	  collection	  to	  measure	  creatinine	  excretion	  plus	  serum	  creatinine	  
5	  
Repeat	  prior	  to	  every	  course	  of	  doxorubicin	  if	  SF	  <30%	  on	  prior	  cycle	  
6	  
Substitute	  CT	  scan	  if	  metal	  in	  endoprothesis	  does	  not	  allow	  for	  MRI	  to	  be	  performed	  
7	  
Perform	  chest	  CT	  or	  bone	  scan	  only	  if	  positive	  for	  metastatic	  disease	  at	  baseline	  
8	  
Pantoprazole	  will	  be	  administered	  either	  on	  cycles	  1	  &	  2	  or	  cycles	  3	  &	  4	  based	  on	  the	  treatment	  arm	  to	  which	  the	  subject	  is	  randomized	  
9	  
Baseline;	  prior	  to	  cisplatin	  on	  day	  1,	  at	  end	  of	  cisplatin	  infusion	  on	  day	  2,	  and	  day	  7;	  prior	  to	  HDMTX	  and	  24	  h	  after	  the	  start	  of	  the	  infusion	  on	  days	  22/23	  &	  
29/30	  (cycles	  1-­‐4);	  pre-­‐op	  and	  day	  1	  of	  cycle	  5.	  A	  separate	  aliquot	  of	  same	  urine	  specimen	  saved	  for	  urine	  creatinine.	  See	  Section	  4.0	  for	  sample	  collection,	  
processing	  and	  storage	  instructions.	  
10	   Baseline;	  prior	  to	  each	  course	  of	  cisplatin	  and	  each	  dose	  of	  HDMTX	  (cycles	  1-­‐4)	  and	  post-­‐treatment	  IF	  serum	  creatinine	  increases	  by	  ≥50%	  compared	  to	  
pretreatment	  value;	  pre-­‐op	  and	  day	  1	  of	  cycle	  5.	  Draw	  simultaneous	  BMP	  with	  serum	  cystatin	  C.	  See	  Section	  4.1	  for	  sample	  collection,	  processing	  and	  
storage	  instructions	  
11	   Serum	  Mg	  and	  creatinine	  and	  spot	  urine	  for	  Mg	  and	  creatinine	  at	  baseline,	  prior	  to	  the	  start	  of	  the	  cisplatin	  infusions	  on	  day	  1	  and	  day	  2	  and	  on	  day	  7	  of	  
cycles	  1-­‐4,	  day	  1	  of	  cycle	  5,	  and	  at	  the	  end	  of	  therapy	  
12	   End	  of	  each	  HDMTX	  infusion	  (cycles	  1-­‐4).	  See	  Section	  4.1	  for	  sample	  collection,	  processing	  and	  storage	  instructions	  
13	   Appendix	  3	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,,	  2013	  
	  
xv  
STUDY	  DIAGRAM	  
	  
	  
4h#MTX#
C#+#PTZ#
12h#MTX#
C#
4h#MTX#
C#
12h#MTX#
C#+#PTZ#
12h#MTX#
C#+#PTZ#
4h#MTX#
C#
12h#MTX#
C#
4h#MTX#
C#+#PTZ#
1#
2#
3#
4#
Cycles#
1#&#2#
Cycles#
3#&#4#
Randomize#
MTX:#
High?dose#methotrexate#by#4h#or#12h#infusion#
C:#
CisplaGn#
PTZ:#
Pantoprazole#
Treatment	  arm	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
1	  
1.	   BACKGROUND	  INFORMATION	  AND	  RATIONALE	  
1.1	   Introduction	  
Conventional	   chemotherapy	   administered	   neoadjuvantly	   (prior	   to	   surgical	   resection	   of	   the	  
primary	  tumor)	  and	  adjuvantly	  after	  tumor	  resection	  improves	  survival	  in	  children,	  adolescents	  
and	  young	  adults	  with	  non-­‐metastatic	  osteosarcoma	  from	  15%	  with	  surgery	  alone	  to	  65%	  by	  
combining	  chemotherapy	  with	  surgery.2,7	  However,	  survival	  has	  not	  improved	  since	  the	  efficacy	  
of	  adjuvant	  chemotherapy	  was	  demonstrated	  in	  the	  mid-­‐1980s,	  as	  illustrated	  in	  Figure	  1.2,8-­‐10	  
Survival	  in	  the	  10	  to	  20%	  of	  patients	  who	  present	  with	  overt	  metastatic	  disease	  at	  diagnosis	  is	  
poor	   despite	   intensive	   treatment,11-­‐13	   and	   the	   subsequent	   development	   of	   overt	   metastatic	  
disease	  is	  the	  primary	  factor	  limiting	  survival	  in	  patients	  who	  present	  with	  localized	  disease.	  
Conventional	   cytotoxic	   chemotherapy	   eradicates	   micrometastatic	   disease	   in	   about	   half	   the	  
patients	  with	  localized	  osteosarcoma,	  but	  the	  most	  effective	  known	  treatment	  regimens	  cause	  
substantial	   acute	   and	   long-­‐term	   toxicity,	   including	   nephrotoxicity,	   cardiotoxicity,	   ototoxicity	  
and	   neurotoxicity.7,14-­‐17	   Improving	   the	   outcome	   for	   patients	   with	   osteosarcoma	   in	   the	   short	  
term	  can	  be	  achieved	  by	  lessening	  the	  toxicity	  of	  standard	  cytotoxic	  treatment	  regimens,	  but	  
we	  must	  also	  identify	  new	  treatment	  targets	  in	  tumors	  cells	  from	  patients	  with	  osteosarcoma	  in	  
order	  to	  develop	  more	  effective	  and	  less	  toxic	  drugs	  for	  this	  disease.	  
	  
1.2	   Compliance	  Statement	  
This	  study	  is	  being	  conducted	  in	  full	  accordance	  with	  all	  applicable	  The	  Children’s	  Hospital	  of	  
Philadelphia	  Research	  Policies	  and	  Procedures	  and	  all	  applicable	  Federal	  and	  State	  laws	  and	  
regulations	  including	  45	  CFR	  46,	  and	  the	  HIPAA	  Privacy	  Rule.	  Any	  episode	  of	  noncompliance	  will	  
be	  documented.	  
The	  investigators	  will	  perform	  the	  study	  in	  accordance	  with	  this	  protocol,	  will	  obtain	  consent	  
and	   assent	   when	   appropriate,	   and	   will	   report	   unexpected	   problems	   in	   accordance	   with	   The	  
Children’s	  Hospital	  of	  Philadelphia	  IRB	  Policies	  and	  Procedures	  and	  all	  Federal	  requirements.	  
	  
Figure	  1	  
Overall	  5-­‐yr	  survival	  for	  osteosarcoma	  diagnosed	  during	  different	  time	  periods.2	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
2	  
Collecting,	  recording,	  and	  reporting	  of	  data	  will	  be	  accurate	  and	  will	  ensure	  the	  privacy,	  health,	  
and	  welfare	  of	  research	  subjects	  during	  and	  after	  the	  study.	  
1.3	   Relevant	  Literature	  and	  Data	  
1.3.1	   Osteosarcoma	  
Osteosarcoma	  is	  the	  most	  common	  type	  of	  bone	  cancer	  in	  children	  and	  adolescents	  under	  the	  
age	  of	  20	  yrs.	  Four	  hundred	  cases	  occur	  annually	  in	  the	  U.S.,	  and	  the	  peak	  incidence	  occurs	  at	  
the	  time	  of	  the	  adolescent	  growth	  spurt.18	  The	  cell	  of	  origin	  for	  osteosarcomas	  is	  believed	  to	  be	  
primitive	   bone-­‐forming	   mesenchymal	   stem	   cells.	   Osteosarcomas	   arise	   primarily	   in	   the	  
metaphyses	  of	  long	  bones	  –	  78%	  in	  the	  lower	  extremities	  (64%	  around	  the	  knee)	  and	  10%	  in	  
the	  humerus.19	  More	  than	  85%	  of	  patients	  present	  with	  localized	  disease.	  The	  most	  common	  
sites	   of	   metastases	   at	   presentation	   are	   the	   lung	   (85%)	   and	   other	   bones	   (20%).19	   Pain	   and	  
swelling	  are	  the	  most	  common	  presenting	  symptoms,	  and	  the	  median	  duration	  of	  symptoms	  
prior	  to	  diagnosis	  is	  69	  days.	  
The	  standard	  management	  of	  osteosarcoma	  starts	  with	  a	  biopsy	  of	  the	  primary	  tumor	  for	  a	  
histological	   diagnosis.	   Suspected	   metastatic	   lesions	   in	   the	   lung	   may	   also	   be	   resected	   at	  
presentation.	   Patients	   then	   receive	   pre-­‐operative	   (neoadjuvant)	   multi-­‐agent	   chemotherapy	  
followed	   by	   surgical	   resection	   of	   the	   primary	   tumor,	   and	   then	   additional	   adjuvant	  
chemotherapy	  for	  a	  total	  of	  6	  to	  8	  months	  of	  treatment.20	  Limb	  salvage	  procedures,	  in	  which	  
the	   tumor	   and	   involved	   bone	   are	   replaced	   by	   an	   endoprosthesis,	   are	   the	   preferred	   surgical	  
approach	   when	   feasible,	   rather	   than	   amputation.	   The	   chemosensitivity	   of	   the	   tumor	   to	   the	  
neoadjuvant	  chemotherapy	  is	  assessed	  in	  the	  resected	  specimen	  by	  microscopically	  quantifying	  
the	  percent	  of	  the	  tumor	  that	  is	  necrotic,	  which	  is	  predictive	  for	  outcome.21	  The	  efficacy	  of	  
adjuvant	   chemotherapy	   for	   localized	   osteosarcoma	   has	   been	   demonstrated	   in	   randomized	  
clinical	  trials.7,22	  
Four	  conventional	  cytotoxic	  anticancer	  drugs	  have	  activity	  in	  osteosarcoma	  (Table	  1).	  Adjuvant	  
regimens	   include	   2,	   3,	   or	   all	   4	   of	   these	   agents.	   A	   meta-­‐analysis	   comparing	   event-­‐free	   and	  
overall	   survival	   for	   regimens	   containing	   2	   drugs	   (n=4),	   3	   drugs	   (n=8)	   or	   4	   drugs	   (n=6)	  
demonstrated	  better	  survival	  for	  3	  drug	  regimens	  than	  regimens	  containing	  2	  drugs	  (HR	  0.70	  
[95%	   CI	   0.68-­‐0.93]	   for	   EFS)	   but	   no	   additional	   advantage	   for	   4	   drug	   regimens.2	   The	   3	   drug	  
combination	   used	   in	   7	   of	   the	   8	   regimens	   in	   this	   analysis	   included	   high-­‐dose	   methotrexate	  
(HDMTX),	  doxorubicin	  and	  cisplatin	  (MAP),	  which	  is	  also	  the	  standard	  regimen	  in	  the	  recently	  
completed	   COG	   trial	   (AOST0331).	   The	   prior	   COG	   trial,	   which	   evaluated	   the	   addition	   of	  
ifosfamide	  and	  MTP-­‐PE	  to	  the	  standard	  MAP	  regimen	  using	  a	  2	  x	  2	  factorial	  design	  failed	  to	  
show	  
a	  
survival	  
advantage	  
for	  
the	  
addition	   of	   ifosfamide,	  
but	   the	   results	   were	  
confounded	  
by	  
a	  
possible	  
interaction	  
between	  
ifosfamide	  
and	  
MTP-­‐PE.23,24	  
A	  
Table	  1	   Response	  rates	  for	  drugs	  used	  to	  treat	  osteosarcoma.2	  
Drug	  
Dose	  Range	  
[mg/m2/course]	  
No.	  of	  
Patients	  
Response	  Rates	  [%]	  
CR	  
PR	  
Doxorubicin	  
35-­‐90	  
108	  
13	  
30	  
Ifosfamide	  
5,000-­‐15,000	  
246	  
12	  
20	  
Methotrexate	  
80-­‐15,000	  
164	  
16	  
16	  
Cisplatin	  
120-­‐625	  
174	  
10	  
16	    


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
3	  
recent	   Italian	   study	   did	   not	   find	   an	   advantage	   to	   adding	   ifosfamide	   to	   MAP.25	   The	   current	  
international	   study,	   which	   recently	   completed	   enrollment,	   is	   evaluating	   the	   utility	   of	   adding	  
ifosfamide	  post-­‐operatively	  in	  patients	  with	  a	  poor	  histological	  response	  (≤90%	  tumor	  necrosis)	  
to	   neoadjuvant	   MAP	   chemotherapy	   and	   adding	   interferon-­‐α	   to	   MAP	   in	   patients	   who	  
experience	   a	   good	   histological	   response.	   Until	   the	   results	   of	   this	   most	   recent	   study	   are	  
available,	  MAP	  (Figure	  2)	  remains	  the	  standard	  of	  care	  for	  osteosarcoma.	  
	  
1.3.2	   Nephrotoxicity	  
Osteosarcoma	  chemotherapy	  regimens	  are	  associated	  with	  acute	  and	  long-­‐term	  nephrotoxicity	  
from	  cisplatin,	  HDMTX,	  and	  ifosfamide.26	  The	  mechanism	  of	  kidney	  injury	  differs	  for	  the	  three	  
drugs.	  MTX	  can	  transiently	  alter	  glomerular	  filtration	  but	  is	  not	  tubulotoxic,	  cisplatin	  directly	  
damages	  renal	  tubular	  epithelium	  resulting	  in	  decreased	  glomerular	  filtration	  and	  electrolyte	  
wasting,	  and	  ifosfamide	  is	  toxic	  to	  renal	  tubules	  and	  primarily	  affects	  the	  proximal	  renal	  tubules	  
but	  can	  also	  alter	  glomerular	  function.	  Hydration,	  alkalinization	  (HDMTX),	  and	  slowing	  the	  dose	  
rate	   (cisplatin)	   can	   lessen	   (but	   not	   prevent)	   nephrotoxicity	   from	   cisplatin	   and	   HDMTX.	   A	  
prospective	   evaluation	   of	   long	   term	   (post-­‐treatment)	   renal	   function	   in	   patients	   with	  
osteosarcoma	   treated	   with	   HDMTX,	   cisplatin	   and	   ifosfamide	   demonstrated	   persistent	  
glomerular	   and	   tubular	   impairment.27	   Half	   had	   a	   reduction	   in	   creatinine	   clearance	   and	  
proteinuria	  and	  25%	  had	  glycosuria	  and	  impaired	  phosphate	  reabsorption.	  
1.3.2.1	   High-­‐Dose	  Methotrexate	  
Methotrexate	   inhibits	   dihydrofolate	   reductase	   (DHFR)	   and	   depletes	   intracellular	   pools	   of	  
tetrahydrofolates,	   which	   are	   required	   for	   the	   synthesis	   of	   thymidine	   and	   purines.	   MTX	   is	  
administered	  in	  high	  doses	  by	  prolonged	  intravenous	  infusion	  because	  prolonged	  exposure	  to	  a	  
cytotoxic	   drug	   concentration	   is	   the	   primary	   determinant	   of	   MTX’s	   antitumor	   effect.28	   High	  
doses	   of	   MTX	   are	   also	   thought	   to	   overcome	   mechanisms	   of	   resistance	   to	   MTX,	   such	   as	  
increased	  expression	  of	  DHFR	  and	  decreased	  expression	  of	  the	  reduced	  folate	  carrier,	  which	  is	  
responsible	  for	  uptake	  of	  the	  drug	  into	  tumor	  cells.	  HDMTX	  can	  also	  enhance	  the	  intracellular	  
formation	  of	  MTX-­‐polyglutamates,	  which	  are	  retained	  in	  cells	  and	  prolong	  the	  drug’s	  effect.28,29	  
HDMTX	   requires	   the	   subsequent	   administration	   of	   leucovorin	   (5-­‐formyl-­‐tetrahydrofolate)	   to	  
rescue	  patients	  from	  the	  potentially	  lethal	  toxic	  effects	  of	  HDMTX.	  Leucovorin	  is	  administered	  
	  
Figure	  2	  
Schema	  of	  the	  MAP	  regimen	  used	  in	  the	  treatment	  of	  osteosarcoma.	  The	  first	  two	  5-­‐week	  cycles	  
are	  administered	  neoadjuvantly.	  Surgery	  is	  performed	  at	  week	  11,	  followed	  by	  4	  additional	  
treatment	  cycles	  (MAP	  x	  2	  and	  MA	  x	  2).	  Numbers	  at	  the	  top	  are	  the	  week	  of	  treatment	  regimen.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
4	  
until	  plasma	  concentrations	  of	  MTX	  fall	  below	  0.1	  
mcM.	  MTX	  is	  eliminated	  by	  renal	  excretion	  –	  more	  
than	  90%	  of	  the	  administered	  dose	  appears	  in	  the	  
urine	   as	   parent	   (unchanged)	   drug.30	   A	   small	  
fraction	   of	   methotrexate	   is	   metabolized	   to	   7-­‐OH-­‐
MTX.6	  
HDMTX	   nephrotoxicity	   is	   dose	   related	   and	   is	  
thought	  to	  result	  from	  precipitation	  of	  MTX	  in	  urine	  in	  renal	  tubules	  as	  a	  result	  of	  MTX’s	  limited	  
solubility	   in	   acidic	   solutions.31-­‐33	   MTX-­‐induced	   nephrotoxicity	   results	   in	   delayed	   excretion	   of	  
MTX,	  sustained	  and	  elevated	  plasma	  MTX	  concentrations,	  which	  may	  not	  be	  effectively	  rescued	  
by	   leucovorin,	   and	   marked	   enhancement	   of	   MTX’s	   other	   systemic	   toxicities,	   such	   as	  
myelosuppression,	   mucositis,	   dermatitis,	   and	   hepatotoxicity.	   The	   incidence	   of	   severe	  
nephrotoxicity	  from	  HDMTX	  in	  osteosarcoma	  is	  1.8%	  and	  in	  4%	  of	  cases	  it	  is	  fatal.32	  Severe	  MTX	  
nephrotoxicity	  is	  a	  medical	  emergency	  that	  requires	  high	  doses	  of	  leucovorin	  to	  protect	  the	  
patient	  and	  glucarpidase	  (carboxypeptidase-­‐G2	  –	  metabolizes	  MTX	  to	  an	  inactive	  metabolite)	  or	  
hemodialysis	  to	  remove	  MTX.	  Less	  severe	  nephrotoxicity	  is	  more	  common34	  and	  requires	  more	  
prolonged	  leucovorin	  rescue,	  which	  can	  compromise	  the	  antitumor	  effect	  of	  MTX.35	  This	  may	  
result	   in	   delays	   in	   subsequent	   therapy,	   thus	   reducing	   dose	   intensity.	   At	   CHOP	   in	   2011,	   240	  
courses	   of	   high	   dose	   methotrexate	   (HDMTX	   ≥1	   g/m2)	   were	   administered	   to	   children	   with	  
osteosarcoma	   (n=15)	   or	   leukemia/lymphoma	   (n=45);	   5%	   of	   courses	   were	   associated	   with	  
delayed	  excretion	  of	  methotrexate,	  likely	  due	  to	  acute	  changes	  in	  renal	  function.	  Infusion	  of	  
HDMTX	  over	  4	  hours	  in	  osteosarcoma	  regimens	  puts	  patients	  at	  higher	  risk	  for	  nephrotoxicity	  
because	  of	  the	  high	  peak	  plasma	  concentrations	  and	  high	  urinary	  concentrations	  of	  the	  drug.	  
Standard	   methods	   to	   prevent	   kidney	   damage	   from	   HDMTX	   include	   vigorous	   hydration	   and	  
alkalinization,	  which	  improves	  MTX	  solubility	  in	  the	  urine	  (Table	  2).6	  At	  a	  pH	  of	  7.0,	  MTX	  (pKa,	  
4.8	  and	  5.7)	  is	  more	  than	  98%	  ionized,	  so	  further	  increases	  in	  the	  pH	  are	  not	  likely	  to	  improve	  
solubility.	  
Table	  2	   pH-­‐dependent	   solubility	   of	   MTX	   and	  
its	  metabolite	  (7-­‐OH-­‐MTX)	  at	  37°C.6	  
	  
Solubility	  [mM]	  
Compound	  
pH	  5.0	  
pH	  6.0	  
pH	  7.0	  
MTX	  
0.97	  
3.5	  
19.6	  
7-­‐OH-­‐MTX	  
0.28	  
0.79	  
3.3	    
	  
	  
Figure	  3	  
Plasma	  (in	  mcM)	  and	  urine	  (in	  mM)	  MTX	  concentrations	  with	  12	  g/m2	  infused	  over	  4	  h.	  Horizontal	  
lines	  represent	  the	  solubility	  limit	  of	  MTX	  at	  a	  pH	  of	  5.0	  and	  7.0.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
5	  
We	  fit	  a	  2-­‐compartment	  pharmacokinetic	  
model	  
to	  
the	  
mean	  
plasma	  
MTX	  
concentrations	  from	  1,045	  infusions	  of	  12	  
g/m2	   over	   4	   h	   (Figure	   3).5	   The	   urinary	  
concentration-­‐time	   profile	   could	   then	   be	  
simulated	  assuming	  that	  the	  rate	  of	  urine	  
production	   was	   equal	   to	   the	   IV	   fluid	  
hydration	   rate	   of	   100	   ml/m2/h	   used	   at	  
CHOP	  with	  the	  equation:	  
CUrine
MTX = Ccentral
MTX •Vcentral •kel
IV!rate
•0.9	  
where	  Curine	  is	  the	  MTX	  in	  urine,	  Ccentral	  is	  
the	   MTX	   concentration	   in	   the	   central	  
compartment,	  Vcentral	  is	  the	  volume	  of	  the	  
central	  
compartment,	  
kel	  
is	  
the	  
elimination	  
rate	  
constant	  
and	  
0.9	  
accounts	  for	  the	  fraction	  of	  the	  MTX	  dose	  excreted	  in	  the	  urine	  unchanged.	  The	  urine	  MTX	  
concentration-­‐time	   profile	   is	   shown	   in	   Figure	   3.	   The	   urine	   MTX	   concentration	   exceeds	   the	  
solubility	  limit	  in	  urine	  at	  a	  pH	  of	  7.0	  for	  4.8	  h,	  putting	  the	  kidneys	  at	  risk	  for	  injury	  from	  MTX	  
precipitation.	  Figure	  4	  shows	  the	  urine	  MTX	  concentration-­‐time	  profiles	  for	  12	  g/m2	  infused	  
over	  4,	  8,	  12	  and	  24	  h.	  With	  the	  longer	  infusion	  durations,	  urine	  MTX	  concentrations	  are	  below	  
the	  solubility	  limit,	  and	  the	  risk	  of	  MTX	  precipitation	  should	  be	  lower.	  
In	  addition	  to	  lowering	  peak	  plasma	  and	  urine	  concentrations	  and	  potentially	  lowering	  the	  risk	  
of	  nephrotoxicity,	  prolonging	  HDMTX	  infusions	  has	  a	  strong	  pharmacological	  rationale	  because	  
the	  duration	  of	  exposure	  to	  a	  cytotoxic	  MTX	  concentration	  is	  the	  primary	  determinant	  of	  the	  
drug’s	  anti-­‐tumor	  effect.28	  Retrospective	  analyses	  from	  European	  studies	  have	  demonstrated	  
an	  association	  between	  high	  peak	  (end	  of	  infusion)	  plasma	  MTX	  concentration	  and	  improved	  
survival.5,36,37	  However,	  the	  role	  of	  peak	  plasma	  MTX	  concentration	  in	  determining	  outcome	  is	  
not	  supported	  by	  the	  observations	  that:	  
• peak	  plasma	  concentrations	  were	  highly	  correlated	  with	  area	  under	  the	  curve	  (AUC)	  and	  
other	   pharmacokinetic	   parameters	   in	   these	   studies,5,36	   making	   difficult	   to	   ascertain	  
whether	  the	  peak	  concentration	  was	  the	  critical	  determinant;	  
• the	   critical	   peak	   MTX	   concentration	   varied	   depending	   on	   the	   dose	   and	   infusion	  
duration.	  For	  12	  g/m2	  of	  MTX	  infused	  over	  4	  h,	  peak	  concentrations	  >1,000	  mcM	  were	  
associated	  with	  better	  survival,	  whereas	  for	  doses	  of	  8-­‐10	  gm/m2,	  the	  cut	  point	  for	  peak	  
concentration	  was	  700	  mcM,	  indicating	  that	  an	  absolute	  peak	  concentration	  is	  not	  the	  
critical	  determinant	  of	  outcome;37	  
• in	   other	   osteosarcoma	   studies,	   no	   relationship	   was	   found	   between	   peak	   MTX	  
concentration	   and	   survival,38-­‐40	   and	   in	   one	   study,	   very	   high	   MTX	   exposures	   were	  
associated	  with	  a	  poorer	  outcome;41	  and	  
	  
Figure	  4	  
Simulations	  of	  urine	  MTX	  concentrations	  with	  
12	  g/m2	  infused	  over	  4,	  8,	  12	  and	  24	  h.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
6	  
• disease-­‐free	   survival	   was	   similar	   for	   7,500	   mg/m2	   of	   MTX	   infused	   over	   6	   h	   and	   690	  
mg/m2	  infused	  over	  42	  h	  in	  a	  randomized	  clinical	  trial	  in	  OS.42	  
These	   observations	   along	   with	   data	   that	   demonstrate	   that	   prolonged	   infusions	   of	   MTX	   are	  
more	  efficacious	  for	  other	  cancers29	  do	  not	  support	  the	  importance	  of	  peak	  concentration	  as	  
the	  critical	  determinant	  of	  HDMTX	  efficacy	  in	  OS.	  
1.3.2.2	   Cisplatin	  
Cisplatin	  is	  an	  inorganic	  compound	  that	  has	  a	  broad	  spectrum	  of	  activity	  in	  cancers	  occurring	  in	  
adults	   and	   children,	   including	   osteosarcoma.	   Its	   mechanism	   of	   action,	   platination	   of	   DNA,	   is	  
analogous	  to	  alkylation	  by	  the	  nitrogen	  mustards	  and	  other	  bifunctional	  alkylating	  agents.	  A	  
reactive	  intermediate,	  which	  is	  formed	  spontaneously	  in	  solution	  through	  aquation	  by	  the	  loss	  
of	   chloride	   (leaving	   group),	   non-­‐discriminately	   forms	   a	   covalent	   bond	   with	   any	   nearby	  
nucleophilic	  group.	  The	  platination	  of	  a	  nucleotide	  in	  DNA	  damages	  the	  DNA	  template	  and	  can	  
lead	  to	  inter-­‐	  and	  intra-­‐strand	  crosslinks.43	  
Cisplatin	  is	  neurotoxic,	  ototoxic,	  highly	  emetogenic	  and	  nephrotoxic,	  and	  these	  toxicities	  can	  
limit	   our	   ability	   to	   administer	   the	   drug	   to	   some	   patients.	   Cisplatin	   nephrotoxicity	   is	   acutely	  
manifested	   as	   lower	   glomerular	   filtration	   rate	   and	   electrolyte	   (magnesium,	   potassium,	  
phosphate)	   wasting	   in	   urine,44	   and	   these	   effects	   appear	   to	   be	   cumulative	   and	   only	   partially	  
reversible.45,46	  The	  mean	  decrease	  in	  glomerular	  filtration	  rate	  for	  a	  dose	  of	  100	  mg/m2	  was	  8%	  
per	  dose	  but	  was	  highly	  variable	  across	  patients	  in	  one	  study.46	  In	  some	  cases,	  the	  severity	  of	  
nephrotoxicity	   compromises	   our	   ability	   to	   administer	   subsequent	   doses	   of	   cisplatin	   and	   can	  
also	   effect	   HDMTX	   elimination	   by	   the	   kidneys.	   Current	   methods	   of	   attenuating	   cisplatin	  
nephrotoxicity	  can	  reduce	  its	  severity	  but	  do	  not	  prevent	  it.	  Intravenous	  fluid	  hydration	  with	  
normal	  saline	  prior	  to	  and	  after	  cisplatin	  infusion	  and	  fractionating	  the	  dose	  over	  several	  days	  
or	  prolonging	  the	  duration	  of	  drug	  infusion	  have	  been	  the	  most	  successful	  methods.47	  The	  role	  
of	  diuretics	  (mannitol	  or	  furosemide)	  remains	  controversial.	  
Cisplatin-­‐related	   renal	   tubular	   and	   vascular	   injury	   is	   a	   complex,	   multi-­‐factorial	   process.44	  
Cisplatin	   uptake	   into	   and	   efflux	   from	   cancer	   cells	   appears	   to	   be	   mediated	   by	   copper	  
transporters	   (CTR1,	   ATP7B).44	   However,	   in	   the	   renal	   tubule,	   the	   polyspecific	   organic	   cation	  
transporters	  (OCTs),	  and	  specifically	  the	  renal	  isoform	  OCT2,	  mediate	  cisplatin	  uptake.48	  OCTs	  in	  
the	  kidney	  mediate	  basolateral	  to	  apical	  transport	  of	  cations,	  and	  on	  the	  apical	  surface,	  cations	  
are	  secreted	  into	  urine	  by	  MATEs	  (multidrug	  and	  toxin	  extrusion	  transporters).49	  Cisplatin	  is	  not	  
transported	   by	   MATEs,	   and	   therefore	   it	   accumulates	   in	   renal	   tubular	   cells.	   Several	   lines	   of	  
evidence	   indicate	   that	   this	   interaction	   of	   cisplatin	   with	   OCT2	   accounts	   for	   its	   selective	  
nephrotoxic	   effects.	   The	   less	   nephrotoxic	   platinum	   analog,	   carboplatin	   is	   not	   an	   OCT2	  
substrate;	  OCT2	  inhibitors	  (e.g.,	  cimetidine)	  can	  block	  cisplatin	  renal	  tubular	  cell	  uptake	  and	  
damage	  in	  vitro	  and	  in	  vivo;	  and	  OCT2-­‐deficient	  animals	  and	  humans	  with	  OCT2	  polymorphisms	  
are	  not	  as	  susceptible	  to	  cisplatin	  nephrotoxicity.48-­‐52	  Tissue	  expression	  of	  OCTs	  is	  limited,	  and	  
they	   are	   not	   expressed	   on	   most	   human	   cancers,	   including	   childhood	   cancers.51,52	   Therefore,	  
inhibiting	  OCT2	  could	  selectively	  rescue	  the	  kidney	  from	  the	  toxic	  effects	  of	  cisplatin	  without	  
interfering	   with	   the	   drug’s	   antitumor	   effect.50	   Patients	   treated	   with	   cisplatin	   may	   also	   be	   at	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
7	  
higher	  risk	  to	  develop	  renal	  cancer	  later	  in	  life,53	  but	  it	  is	  not	  clear	  whether	  this	  is	  related	  to	  
cisplatin	  accumulation	  in	  the	  renal	  tubule	  cells.	  
Cisplatin	  pharmacokinetics	  is	  difficult	  to	  quantify	  because	  it	  is	  chemically	  unstable.	  The	  parent	  
drug	  spontaneously	  forms	  a	  reactive	  aquated	  intermediate	  that	  rapidly	  reacts	  with	  a	  nearby	  
nucleophilic	  group.	  In	  plasma,	  the	  reactive	  intermediates	  react	  with	  plasma	  proteins	  forming	  a	  
covalent	  bond,	  which	  inactivates	  the	  drug.	  Protein	  binding	  increases	  over	  time,	  reflecting	  the	  
non-­‐reversible	  nature	  of	  the	  protein	  binding.	  The	  platinated	  proteins	  are	  slowly	  removed	  from	  
circulation	  and	  degraded,	  which	  releases	  platinum	  in	  a	  form	  that	  is	  no	  longer	  active.	  Studies	  of	  
cisplatin	   pharmacokinetics	   (PK)	   have	   measured	   free	   (non-­‐protein	   bound)	   and	   total	   platinum	  
(bound	  +	  free)	  using	  atomic	  absorption	  spectroscopy.	  The	  free	  form	  of	  platinum,	  which	  includes	  
both	   active	   drug	   species	   and	   inactive	   forms,	   has	   a	   biexponential	   plasma	   concentration-­‐time	  
curve	   with	   half-­‐lives	   of	   20	   min	   and	   >24	   h	   in	   children.54,55	   The	   former	   likely	   represents	   the	  
disappearance	  of	  the	  active	  species	  of	  the	  drug,	  and	  the	  latter,	  more	  prolonged	  half-­‐life	  likely	  
reflects	  the	  disappearance	  of	  inactive,	  non-­‐protein	  bound	  forms.	  Although	  renal	  excretion	  is	  the	  
primary	   route	   of	   platinum	   elimination,	   it	   accounts	   for	   only	   27%	   of	   total	   clearance,55	   and	   a	  
substantial	  fraction	  of	  platinum	  in	  urine	  is	  inactivated	  before	  it	  is	  excreted.	  Therefore,	  renal	  
excretion	  is	  not	  the	  major	  route	  of	  elimination	  for	  the	  active	  species	  of	  the	  drug	  as	  evidenced	  
by	  the	  fact	  that	  cisplatin	  clearance	  in	  an	  anephric	  patient	  was	  not	  substantially	  different	  than	  
that	   of	   patients	   with	   normal	   renal	   function.56	   Co-­‐administration	   of	   an	   OCT2	   inhibitor	   with	  
cisplatin	  should	  not	  interfere	  with	  cisplatin	  clearance,	  because	  cisplatin	  is	  not	  secreted	  after	  it	  is	  
taken	  up	  into	  renal	  tubular	  cells	  (see	  above)	  and	  because	  renal	  excretion	  of	  the	  active	  forms	  of	  
the	  drug	  is	  not	  a	  major	  route	  of	  elimination.	  
1.3.3	   Biomarkers	  of	  Acute	  Kidney	  Injury	  
Current	  clinical	  laboratory	  tests	  of	  renal	  function	  in	  children	  with	  cancer	  do	  not	  detect	  early,	  
subtle	  changes	  in	  glomerular	  filtration	  rate	  (GFR)	  or	  renal	  tubular	  function.	  Limitations	  of	  serum	  
creatinine	  (sCr)	  as	  a	  marker	  of	  GFR57	  include:	  
• Non-­‐renal	   factors	   (diet,	   muscle	   mass,	   tubular	   secretion)	   can	   alter	   the	   sCr	   but	   do	   not	  
affect	  GFR,	  
• sCr	  can	  be	  within	  the	  normal	  range	  with	  a	  GFR	  as	  low	  as	  40	  mL/min/1.73	  m2,	  
• sCr	  is	  imprecise	  in	  small	  children	  and	  infants	  because	  it	  is	  rounded	  to	  the	  first	  decimal	  
place,	  resulting	  in	  a	  significant	  potential	  error	  in	  estimating	  creatinine	  clearance	  (eCrCL),	  
• a	  recent	  change	  in	  the	  method	  used	  to	  quantify	  sCr	  has	  invalidated	  formulas	  used	  for	  
eCrCL.58	  
More	  sensitive	  and	  specific	  biomarkers	  to	  accurately	  quantify	  small	  changes	  GFR	  and	  to	  rapidly	  
detect	   renal	   tubular	   damage	   are	   being	   developed.	   These	   biomarkers	   may	   be	   useful	   for	  
detecting	   and	   monitoring	   renal	   toxicity	   of	   nephrotoxic	   anticancer	   drugs,	   such	   as	   MTX	   and	  
cisplatin.	   We	   plan	   to	   use	   a	   panel	   of	   new	   biomarkers	   (described	   below)	   in	   combination	   with	  
traditional	  laboratory	  tests	  of	  renal	  function	  to	  assess	  the	  impact	  of	  altering	  the	  schedule	  of	  
HDMTX	  and	  adding	  pantopazole	  as	  a	  cisplatin	  rescue	  agent	  on	  nephrotoxicity	  of	  these	  drugs	  on	  
this	  trial.	  These	  new	  biomarkers	  will	  include:	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
8	  
• Cystatin	   C	  is	  a	  13kDa	  cysteine	  protease	  inhibitor	  produced	  by	  all	  nucleated	  cells.	  It	  is	  
freely	   filtered,	   reabsorbed	   and	   metabolized	   in	   proximal	   tubules	   but	   not	   normally	  
secreted	   in	   urine	   by	   the	   kidney.	   Serum	   cystatin	   C	   is	   used	   to	   estimate	   GFR	   and	   urine	  
cystatin	  C	  is	  a	  marker	  of	  tubular	  damage.	  Cystatin	  C	  is	  not	  influenced	  by	  muscle	  mass,	  
and	  it	  has	  improved	  temporal	  discrimination	  in	  detection	  of	  kidney	  injury	  compared	  to	  
sCr.	   Cystatin	   C	   is	   measured	   by	   a	   FDA	   approved	   nephelometry	   method	   in	   clinical	  
laboratories.59-­‐68	  CrCl	  can	  be	  estimated	  from	  sCr	  using	  the	  Schwartz	  formula	  and	  GFR	  is	  
calculated	   from	   a	   sCr	   and	   Cystatin	   C	   based	   formula	   developed	   in	   the	   Children	   with	  
Chronic	  Kidney	  Disease	  Cohort:58	  	  
CrCL=0.413•[height	  (cm)]/sCr	  
	  
Chemotherapy	  induced	  renal	  tubular	  dysfunction	  manifests	  as	  renal	  wasting	  of	  minerals	  and	  
electrolytes.	   In	   current	   clinical	   practice,	   renal	   tubular	   dysfunction	   is	   diagnosed	   when	   serum	  
electrolyte	   concentrations	   fall	   below	   normal	   and	   require	   oral	   supplementation.	   Sensitive	  
biomarkers	  of	  renal	  tubular	  damage	  or	  dysfunction	  could	  detect	  toxicity	  earlier	  and	  contribute	  
to	  the	  evaluation	  of	  strategies	  to	  attenuate	  nephrotoxicity.	  New	  biomarkers	  of	  renal	  tubular	  
damage	  or	  dysfunction	  include:	  
§ Kidney	   Injury	   Molecule-­‐1	   (KIM-­‐1)	   is	   a	   50	   kDa	   trans-­‐membrane	   protein	   expressed	   on	  
proximal	   tubule	   cells.	   Urine	   KIM-­‐1	   is	   a	   marker	   of	   proximal	   tubular	   damage	   that	   may	  
discriminate	  pre-­‐renal	  azotemia	  from	  ischemia.	  	  Urine	  concentrations	  are	  not	  effected	  
by	  chronic	  kidney	  disease.	  ELISA	  kits	  are	  commercially	  available	  for	  research,	  and	  clinical	  
laboratory	  micro-­‐bead	  assays	  are	  under	  development.69-­‐73	  
§ Neutrophil	   Gelatinase-­‐Associated	   Lipocalin	   (NGAL)	   is	   a	   25	   kDa	   protein	   expressed	   in	  
intestine	  lung,	  liver,	  and	  renal	  tubular	  epithelial	  cells.	  Elevations	  in	  plasma	  NGAL	  occur	  
promptly	   after	   renal	   ischemic	   or	   tissue	   injury.	   Plasma	   and	   urine	   NGAL	   are	   early	  
predictors	  of	  acute	  kidney	  injury	  (AKI)	  and	  may	  discriminate	  pre-­‐renal	  from	  intrinsic	  AKI.	  
ELISA	   kits	   are	   commercially	   available	   for	   research	   applications	   and	   clinical	   laboratory	  
assays	  are	  available.74-­‐85	  
§ N-­‐acetyl-­‐β-­‐D-­‐glucosamidase	  (NAG)	  is	  a	  130	  kD	  enzyme	  within	  lysomomes	  of	  proximal	  
tubular	  cells	  of	  the	  kidney.	  Urinary	  NAG	  concentrations	  are	  increased	  during	  oxidative	  
stress	   and	   renal	   tubular	   damage	   including	   from	   cisplatin	   and	   gentamicin.52,86	   Urinary	  
NAG	   increases	   in	   the	   presence	   of	   oxidative	   stress	   and	   proximal	   tubular	   damage	  
regardless	  of	  development	  of	  AKI.	  
Prospective	  systematic	  evaluation	  of	  glomerular	  and	  tubular	  function	  using	  these	  new,	  more	  
sensitive	  biomarkers	  should	  allow	  us	  to	  detect	  more	  subtle	  changes	  in	  GFR	  and	  tubular	  function	  
and	  assess	  the	  impact	  of	  the	  interventions	  to	  attenuate	  HDMTX	  and	  cisplatin	  nephrotoxicity.	  
GFR = 39.1•
Hgt
Crserum
 
 
 
 
 
 
0.516 1.8
CyC
 
 
 
 
 
 
0.294
30
BUNserum
 
 
 
 
 
 
0.169
1.099
[
]
male Hgt
1.4
 
 
 
 
 
 
0.188


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
9	  
The	  fractional	  excretion	  of	  magnesium	  will	  also	  be	  monitored	  during	  cisplatin	  administration	  to	  
assess	  renal	  tubular	  function.	  FEMg	  =	  (100•(Urine	  Mg	  •	  Serum	  Cr))/(0.7•Serum	  Mg	  •	  Urine	  Cr).	  
Multiplying	  serum	  Mg	  by	  0.7	  accounts	  for	  30%	  protein	  binding	  to	  albumin.	  
1.3.4	   Ototoxicity	  
Cisplatin	   causes	   dose-­‐	   and	   schedule-­‐dependent	   permanent,	   sensorineural	   hearing	   loss	   by	  
damaging	   cochlear	   hair	   cells.87	   Initially,	   osteosarcoma	   patients	   experience	   high	   frequency	  
hearing	   loss	   at	   4	   to	   8	   kHz	   at	   cumulative	   doses	   exceeding	   300	   mg/m2,	   but	   hearing	   at	   lower	  
frequencies	  becomes	  impaired	  as	  the	  cumulative	  dose	  increases,	  and	  a	  substantial	  fraction	  of	  
patients	   are	   affected.14,88-­‐90	   Younger	   patients	   are	   at	   greater	   risk	   for	   hearing	   loss	   from	  
cisplatin.91,92	  With	  long-­‐term	  follow-­‐up,	  there	  may	  be	  worsening	  or	  progression	  of	  hearing	  loss	  
at	  lower	  frequencies.93	  
Fractionating	  the	  cisplatin	  dose	  appears	  to	  lessen	  the	  risk	  of	  ototoxicity,88	  but	  more	  effective	  
strategies	  to	  prevent	  damage	  to	  the	  hair	  cells	  are	  needed.	  Sodium	  thiosulfate	  is	  being	  tested	  as	  
a	  rescue	  agent	  in	  an	  ongoing	  COG	  trial,	  but	  thiosulfate	  is	  a	  non-­‐selective	  rescue	  agent	  and	  can	  
interfere	   with	   the	   anti-­‐tumor	   effect	   of	   cisplatin.	   Cochlear	   hair	   cells	   also	   express	   OCT2,	   and	  
inhibition	  of	  OCT2	  during	  cisplatin	  therapy	  may	  selectively	  block	  the	  uptake	  of	  cisplatin	  into	  hair	  
cells	  without	  impacting	  the	  drug’s	  anti-­‐tumor	  effect.48,50	  
1.3.5	   Pantoprazole	  
Pantoprazole	  (Figure	  5)	  is	  a	  proton	  pump	  inhibitor	  (PPI)	  
that	  blocks	  gastric	  acid	  secretion	  by	  irreversibly	  binding	  
to	  the	  proton	  pump.94	  It	  is	  indicated	  for	  treatment	  of	  
GERD	   with	   erosive	   esophagitis	   and	   hypersecretion	  
associated	  
with	  
Zollinger-­‐Ellison	  
syndrome.	  
Pantoprazole	  
is	  
commercially	  
available	  
in	  
an	  
intravenous	   formulation	   that	   can	   be	   administered	   as	  
an	  intermittent	  bolus	  or	  continuous	  infusion.	  
Pantoprazole	  and	  other	  PPIs	  are	  potent	  OCT2	  inhibitors	  (Figure	  6).1	  The	  IC50	  for	  pantoprazole	  
inhibition	  of	  OCT2	  is	  2.8	  mcM	  and	  the	  Cmax	  of	  the	  drug	  in	  children	  after	  0.8	  mg/kg	  (equivalent	  to	  
40	  mg	  in	  adults)	  was	  15	  mcM	  and	  after	  a	  dose	  of	  1.6	  mg/kg	  (equivalent	  to	  80	  mg	  in	  adults)	  was	  
27	  mcM,3	  which	  would	  inhibit	  OCT2	  by	  >80%.1	  
Cimetidine	  (histamine	  H2-­‐receptor	  antagonist)	  also	  inhibits	  
OCT2.	  In	  a	  small	  study	  of	  9	  patients	  with	  testicular	  cancer	  
treated	   with	   cisplatin	   (20	   mg/m2/d	   x	   5	   days),	   cimetidine	  
plus	  
verapamil	  
appeared	  
to	  
prevent	  
cisplatin	  
nephrotoxicity	   (assessed	   as	   change	   in	   effective	   renal	  
plasma	   flow	   and	   GFR)	   compared	   to	   a	   similar	   historical	  
control	   population	   (n=9)	   treated	   with	   cisplatin	   alone	  
(Figure	   7).4	   Cimetidine	   does	   not	   interfere	   with	   the	  
anticancer	   effect	   of	   cisplatin	   in	   vitro	   and	   in	   vivo	   in	   an	  
osteosarcoma	  cell	  line,	  indicating	  that	  uptake	  of	  cisplatin	  
	  
Figure	  5	   Structure	  of	  pantoprazole.	    
Figure	  6	   Inhibition	  of	  OCT2	  by	  panto-­‐
prazole.1	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
10  
into	  tumor	  cells	  is	  not	  mediated	  by	  OCT2	  in	  osteosarcoma.95	  Pretreatment	  of	  human	  tumor	  
cells	  with	  a	  proton	  pump	  inhibitor	  enhanced	  the	  cytotoxic	  effect	  of	  multiple	  anti-­‐cancer	  drugs	  
including	  cisplatin,	  and	  omeprazole	  pretreatment	  in	  mice	  harboring	  human	  tumor	  xenografts	  
induced	  sensitivity	  of	  the	  tumors	  to	  cisplatin.96,97	  
The	  PK	  and	  tolerability	  of	  single	  intravenous	  doses	  of	  0.8	  and	  1.6	  mg/kg	  of	  pantoprazole	  have	  
been	  studied	  in	  children.3	  The	  Cmax	  and	  AUC	  of	  IV	  pantoprazole	  at	  0.8	  and	  1.6	  mg/kg	  in	  children	  
aged	  2	  to	  16	  years	  were	  similar	  to	  these	  PK	  parameters	  in	  adults	  at	  40	  and	  80	  mg	  (Figure	  8).	  The	  
clearance	  of	  pantoprazole	  in	  children	  was	  0.2	  L/h/kg	  and	  the	  half-­‐life	  was	  1.2	  h.	  Although	  the	  
t1/2	   is	   short,	   it	   pharmacodynamic	   effect	   is	   more	   long	   lasting.94	   PPIs	   interfere	   with	   MTX	  
excretion,98	  but	  by	  20	  days	  
after	  the	  last	  cisplatin	  dose	  
when	   HDMTX	   is	   adminis-­‐
tered,	  pantoprazole	  will	  be	  
completely	  cleared.	  
Pantoprazole	   is	   primarily	  
metabolized	   by	   CYP2C19,	  
which	   is	   genetically	   poly-­‐
morphic.	   CYP2C19	   activity	  
is	   absent	   in	   3%	   of	   whites	  
and	   20%	   of	   Asians,	   and	  
children	  
with	  
CYP2C19	  
polymorphisms	  
have	  
slower	  
clearance	  
and	  
longer	  t1/2s.3	  
	  
Figure	  7	   Change	  in	  GFR	  10	  and	  21	  days	  after	  100	  mg/m2	  cisplatin	  administered	  over	  5	  days	  without	  (left)	  and	  
with	  cimetidine	  and	  verapamil.	    
	  
Figure	  8	   PK	  of	  intravenous	  pantoprazole	  in	  children	  (2-­‐16	  yrs)	  at	  doses	  of	  
0.8	  and	  1.6	  mg/kg	  compared	  to	  adults	  at	  20,	  40	  and	  80	  mg.3,4	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
11  
The	  single	  IV	  doses	  of	  pantoprazole	  (0.8	  and	  1.6	  mg/kg)	  were	  well	  tolerated.	  Three	  adverse	  
events	   occurred	   in	   18	   patients,	   all	   of	   which	   were	   mild	   and	   judged	   to	   be	   unrelated	   to	  
pantoprazole.3	   The	   most	   common	   toxicities	   associated	   with	   PPIs	   are	   diarrhea,	   nausea,	  
headaches,	  abdominal	  pain,	  fatigue	  and	  dizziness.94	  
1.3.6	   Serum	  biomarkers	  for	  osteosarcoma	  
Serum	  biomarkers	  for	  diagnosing	  osteosarcoma,	  for	  monitoring	  response	  to	  treatment,	  and	  for	  
detecting	  disease	  recurrence	  would	  be	  valuable	  for	  the	  clinical	  management	  of	  the	  disease	  and	  
as	   endpoints	   for	   clinical	   trials.	   Osteosarcoma	   is	   a	   matrix-­‐producing	   tumor	   and	   radiographic	  
response	  (tumor	  shrinkage)	  to	  neoadjuvant	  chemotherapy	  may	  not	  reflect	  the	  extent	  of	  tumor	  
necrosis,	  histologically,	  after	  neoadjuvant	  chemotherapy.	  
Elevated	  serum	  alkaline	  phosphatase	  (AP)	  is	  an	  independent	  prognostic	  factor	  predicting	  for	  
poor	  outcome	  in	  osteosarcoma,25,99	  but	  serum	  AP	  may	  be	  derived	  from	  other	  organs	  and	  is	  
therefore	  not	  specific.	  Bone-­‐specific	  AP	  (BSAP),	  which	  is	  the	  isoform	  of	  AP	  derived	  from	  bone,	  is	  
a	  more	  specific	  measure	  of	  osteoblastic	  activity.100-­‐102	  BSAP	  is	  elevated	  in	  patients	  with	  OS	  and	  
is	  correlated	  with	  tumor	  burden.102,103	  However,	  BSAP	  is	  also	  higher	  in	  adolescents	  reflecting	  
periods	  of	  bone	  growth	  during	  the	  adolescent	  growth	  spurt.	  We	  will	  investigate	  the	  potential	  
role	   of	   BSAP	   as	   a	   potential	   biomarker	   for	   OS	   by	   measuring	   serum	   levels	   at	   diagnosis,	   after	  
neoadjuvant	  chemotherapy,	  after	  surgical	  resection,	  and	  at	  the	  end	  of	  treatment.	  
1.3.7	   Study	  Rationale	  
Current	  osteosarcoma	  treatment	  regimens	  include	  drugs	  that	  are	  nephrotoxic	  and	  ototoxic,	  and	  
the	  damage	  to	  kidneys	  and	  cochlear	  hair	  cells	  may	  be	  irreversible.	  Preventing	  these	  toxicities	  
will	  improve	  the	  outcome	  in	  long-­‐term	  survivors	  and	  may	  also	  prevent	  short-­‐term	  treatment	  
delays	  and	  dose	  reductions	  that	  can	  compromise	  the	  efficacy	  of	  the	  treatment	  regimen	  and	  
allow	   for	   administration	   of	   higher	   cumulative	   doses	   of	   cisplatin.	   This	   pilot	   study	   evaluates	  
pharmacologically-­‐based	  approaches	  to:	  
• prevent	   the	   nephrotoxic	   effect	   of	   HDMTX	   by	   prolonging	   the	   infusion	   duration	   and	  
thereby	  lowering	  the	  risk	  of	  drug	  precipitation	  in	  renal	  tubules,	  and	  
• selectively	  block	  the	  uptake	  of	  cisplatin	  into	  renal	  tubular	  cells	  and	  cochlear	  hair	  cells	  by	  
inhibiting	   the	   organic	   cation	   transporter	   2	   with	   the	   proton	   pump	   inhibitor,	  
pantoprazole.	  
A	  randomized	  cross-­‐over	  design,	  in	  which	  patients	  serve	  as	  their	  own	  control,	  will	  be	  used	  to	  
maximize	   the	   potential	   to	   detect	   differences	   in	   markers	   of	   acute	   kidney	   injury	   (NAG,	   NGAL,	  
KIM-­‐1,	  cystatin	  C)	  and	  audiograms	  resulting	  from	  altering	  the	  infusion	  duration	  of	  HDMTX	  and	  
from	  administering	  pantoprazole	  with	  cisplatin.	  
Two	   infusion	   durations	   of	   HDMTX	   will	   be	   studied	   –	   the	   standard	   4	   h	   duration	   and	   more	  
prolonged	  12	  h	  infusion	  duration.	  Leucovorin	  rescue	  will	  commence	  24	  h	  from	  the	  start	  of	  the	  
infusion.	  The	  HDMTX	  infusions	  will	  be	  divided	  into	  2	  blocks	  (cycles	  1	  &	  2,	  cycles	  3	  &	  4,	  see	  
Figure	  2),	  each	  of	  which	  include	  4	  HDMTX	  infusions.	  The	  order	  of	  infusion	  duration	  studied	  in	  
each	  block	  will	  be	  randomly	  determined.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
12  
Pantoprazole	   will	   either	   be	   administered	   with	  
the	  first	  two	  courses	  of	  cisplatin	  during	  cycles	  1	  
&	  2	  or	  with	  the	  third	  and	  fourth	  courses	  during	  cycles	  3	  &	  4,	  and	  the	  order	  will	  be	  randomly	  
determined.	  Cisplatin	  is	  administered	  as	  a	  60	  mg/m2	  dose	  infused	  over	  4	  h	  daily	  x	  2	  d,	  and	  
Figure	  9	  is	  a	  PK	  simulation	  showing	  the	  amount	  of	  active	  drug	  in	  the	  body	  (assuming	  a	  half-­‐life	  
of	   20	   min)	   and	   the	   percent	   of	   the	   total	   dose	   cleared.	   At	   steady	   state,	   7.2	   mg/m2	   of	   active	  
cisplatin	  is	  in	  the	  body,	  and	  by	  the	  end	  of	  the	  4	  h	  infusion,	  88%	  of	  the	  total	  dose	  has	  been	  
cleared.	  Therefore,	  adequate	  concentrations	  to	  inhibit	  OCT2	  should	  be	  present	  for	  at	  least	  4	  h	  
to	  prevent	  cisplatin	  nephrotoxicity	  and	  ototoxicity.	  Pantoprazole	  has	  a	  short	  half-­‐life	  (1.2	  h),3	  
and	   when	   administered	   at	   a	   dose	   of	   1.6	   mg/kg	   as	   a	   standard	   15	   min	   infusion	   (Figure	   10),	  
plasma	   pantoprazole	   concentration	   by	   the	   end	   of	   the	   4	   h	   cisplatin	   infusion	   is	   1.3	   mcM.	   By	  
administering	  the	  same	  dose	  as	  a	  0.3	  mg/kg	  loading	  dose	  infused	  over	  15	  min	  followed	  by	  a	  4	  h	  
infusion	   of	   the	   remaining	   1.3	   mg/kg,	   plasma	   pantoprazole	   concentration	   is	   5.9	   mcM	   at	   4	   h	  
(Figure	  10).	  
The	  primary	  endpoint	  to	  assess	  nephrotoxicity	  will	  be	  the	  AKI	  biomarkers	  (cystatin	  C,	  KIM-­‐1,	  
NGAL,	  NAG)	  described	  in	  Section	  1.3.3.	  This	  will	  allow	  us	  to	  assess	  each	  dose	  of	  HDMTX	  and	  
each	   course	   of	   cisplatin	   independently.	   Standard	   renal	   function	   tests	   (urinalysis,	   serum	  
creatinine,	  creatinine	  clearance,	  and	  fractional	  excretion	  of	  magnesium)	  will	  also	  be	  monitored.	  
Audiograms	  will	  be	  performed	  after	  each	  course	  of	  cisplatin	  to	  assess	  ototoxicity.	  Radiographic	  
response	   and	   histological	   response	   in	   the	   resected	   tumor	   specimen	   after	   neoadjuvant	  
chemotherapy	  will	  be	  assessed	  to	  ensure	  changes	  in	  HDMTX	  infusion	  duration	  and	  the	  addition	  
of	   pantoprazole	   to	   cisplatin	   do	   not	   alter	   the	   efficacy	   of	   chemotherapy.	   The	   toxicity	   of	   the	  
regimen	   will	   be	   closely	   monitored,	   and	   we	   will	   pilot	   a	   disease-­‐specific	   patient	   reported	  
outcome	  survey	  to	  assess	  the	  impact	  of	  tumor-­‐related	  and	  drug	  toxicity-­‐related	  symptoms.	  
	  
	  
	  
Figure	  9	   Amount	  of	  cisplatin	  in	  the	  body	  (blue)	  and	  
cumulative	   percent	   of	   the	   dose	   cleared	  
(orange)	  for	  a	  60	  mg/m2	  dose	  infused	  over	  
4	  h.	  
	  
Figure	  10	   Plasma	   concentration-­‐time	   profile	   of	  
1.6	   mg/kg	   pantoprazole	   infused	   over	  
15	  min	  (blue)3	  or	  administered	  as	  a	  0.3	  
mg/kg	   loading	   dose	   over	   15	   min	  
followed	  by	  1.3	  mg/kg	  infused	  over	  4	  h	  
(green).	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
13  
2.	   STUDY	  OBJECTIVES	  
The	   primary	   objective	   is	   to	   identify	   more	   rational,	   pharmacologically-­‐based	   drug	   delivery	  
approaches	  to	  prevent	  nephrotoxicity	  from	  HDMTX	  and	  cisplatin	  and	  ototoxicity	  from	  cisplatin	  
in	  patients	  with	  OS	  by:	  
• comparing	  biomarkers	  of	  acute	  kidney	  injury	  (KIM-­‐1,	  NAG,	  NGAL),	  glomerular	  function	  
(cystatin	  C,	  creatinine)	  and	  plasma	  and	  urine	  MTX	  concentrations	  after	  12	  g/m2	  of	  MTX	  
infused	  over	  4	  and	  12	  hours,	  
• comparing	  biomarkers	  of	  acute	  kidney	  injury	  (KIM-­‐1,	  NAG,	  NGAL)	  and	  glomerular	  and	  
tubular	   function	   (cystatin	   C,	   creatinine,	   FEMg)	   after	   cisplatin	   administered	   with	   and	  
without	  the	  OCT2	  inhibitor,	  pantoprazole,	  and	  
• comparing	  hearing	  loss	  (audiograms)	  after	  cisplatin	  administered	  with	  and	  without	  the	  
OCT2	  inhibitor,	  pantoprazole.	  
Secondary	  objectives	  include:	  
• Comparing	  the	  incidence	  and	  severity	  of	  common	  toxicities	  from	  cisplatin/doxorubicin	  
administered	  with	  and	  without	  pantoprazole	  and	  from	  HDMTX	  administered	  as	  a	  4	  and	  
12	  h	  infusion,	  
• comparing	  radiographic	  response	  (log	  ratio	  of	  tumor	  volumes)	  and	  histological	  response	  
(percent	  tumor	  necrosis)	  to	  two	  cycles	  (10	  weeks)	  of	  neoadjuvant	  chemotherapy	  for	  the	  
two	  infusion	  durations	  of	  HDMTX	  and	  for	  cisplatin	  with	  and	  without	  pantoprazole,	  
• correlating	   urinary	   biomarkers	   of	   AKI	   and	   GFR	   estimated	   from	   serum	   cystatin	   C	   to	  
standard	  measures	  of	  renal	  function	  (serum	  creatinine,	  urinalysis,	  estimated	  creatinine	  
clearance,	  FEMg),	  
• building	   a	   tissue	   microarray	   from	   biopsy,	   primary	   resection	   and	   resected	   metastatic	  
tumors	  to	  evaluate	  the	  expression	  of	  proteins	  that	  are	  responsible	  for	  resistance	  to	  the	  
drugs	  in	  the	  MAP	  regimen	  and	  to	  assess	  expression	  of	  proteins	  that	  are	  targeted	  by	  new	  
anticancer	  drugs	  under	  development	  for	  childhood	  cancers,	  	  
• evaluating	  serum	  BSAP	  as	  a	  potential	  biomarker	  for	  OS,	  
• monitoring	   nutritional	   status	   (weight,	   arm	   circumference,	   skin	   fold	   thickness,	   pre-­‐
albumin)	  in	  patients	  receiving	  the	  MAP	  chemotherapy	  regimen,	  and	  
• piloting	   an	   osteosarcoma-­‐specific	   patient	   reported	   outcomes	   survey	   to	   assess	   the	  
incidence	  and	  severity	  of	  tumor-­‐related	  and	  treatment-­‐related	  symptoms.	  
3.	   INVESTIGATIONAL	  PLAN	  
3.1	   General	  Schema	  of	  Study	  Design	  
Patients	   with	   previously	   untreated	   osteosarcoma	   will	   receive	   6	   cycles	   of	   the	   standard	   MAP	  
(Methotrexate,	  Adriamycin	  [doxorubicin],	  cisPlatin)	  chemotherapy	  regimen.	  The	  first	  two	  cycles	  
will	   be	   administered	   neoadjuvantly	   followed	   by	   surgical	   resection	   of	   the	   primary	   tumor	   if	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
14  
feasible,	   and	   then	   2	   additional	   cycles	   of	   MAP	   followed	   by	   2	   cycles	   of	   MA	   (Figure	   2).	   A	  
randomized,	  2	  x	  2	  factorial,	  crossover	  design	  (see	  Study	  Diagram)	  will	  be	  used	  to:	  
• study	  the	  effect	  of	  prolonging	  the	  methotrexate	  infusion	  duration	  from	  4	  h	  to	  12	  h	  on	  
nephrotoxicity	  as	  measured	  by	  biomarkers	  of	  acute	  renal	  injury	  and	  
• study	   the	   effect	   of	   the	   OCT2	   inhibitor,	   pantoprazole,	   on	   cisplatin	   nephrotoxicity	   as	  
measured	  by	  biomarkers	  of	  acute	  renal	  injury	  and	  on	  ototoxicity	  as	  assessed	  on	  serial	  
audiograms.	  
Each	   patient	   will	   serve	   as	   his/her	   own	   control,	   and	   24	   evaluable	   patients	   will	   be	   studied.	  
Randomization	   to	   one	   of	   4	   treatment	   arms	   (see	   Study	   Diagram)	   will	   be	   performed	   after	  
enrollment	   onto	   the	   trial	   and	   prior	   to	   the	   start	   of	   neoadjuvant	   chemotherapy.	   The	   use	   of	  
sensitive,	   short-­‐term	   biomarkers	   as	   endpoints	   to	   assess	   the	   effect	   of	   these	   interventions,	  
continuous	   (rather	   than	   categorical)	   endpoints	   analyzed	   by	   dose,	   course	   or	   treatment	   cycle	  
rather	   than	   by	   patient,	   and	   a	   cross-­‐over	   design	   that	   allows	   patients	   to	   serve	   as	   their	   own	  
controls	  will	  minimize	  the	  number	  of	  subjects	  and	  time	  required	  to	  complete	  this	  pilot	  study,	  
increase	  the	  statistical	  power,	  and	  maximize	  the	  amount	  of	  data	  collected.	  
3.1.1	   Screening,	  Diagnosis,	  and	  Staging	  
Patients	  with	  osteosarcoma	  typically	  present	  to	  the	  orthopedic	  or	  oncology	  clinical	  service	  with	  
a	   history	   of	   pain	   and	   swelling	   at	   the	   primary	   tumor	   site	   and	   radiographic	   evidence	   of	   a	  
destructive	  bone	  lesion.	  The	  standard	  of	  care	  at	  CHOP	  is	  to	  perform	  an	  open	  biopsy	  to	  ensure	  
that	  an	  adequate	  amount	  of	  viable	  tumor	  tissue	  is	  obtained	  to	  make	  a	  diagnosis.	  Histological	  
diagnosis	  is	  based	  on	  morphology	  and	  the	  presence	  of	  osteoid	  (unmineralized	  bone	  matrix).	  
There	  are	  no	  specific	  immunohistochemical	  stains	  that	  can	  be	  used	  to	  confirm	  the	  diagnosis	  of	  
osteosarcoma.	  
The	   standard	   radiographic	   staging	   for	   osteosarcoma	   to	   assess	   tumor	   extent	   in	   the	   involved	  
bone	   and	   to	   document	   the	   presence	   of	   overt	   metastatic	   disease	   in	   the	   lungs	   or	   skeleton	  
includes:	  
• AP	  and	  lateral	  radiographic	  of	  the	  entire	  involved	  bone	  
• MRI	  with	  contrast	  of	  the	  entire	  involved	  bone,	  including	  joints	  proximal	  and	  distal	  to	  the	  
tumor	  (e.g.,	  for	  a	  distal	  femoral	  tumor,	  the	  scan	  should	  include	  the	  entire	  femur	  with	  
the	  hip	  and	  knee	  joints)	  
• Chest	  CT	  scan	  
• 
99mTc-­‐MDP	  Bone	  scan	  OR	  18FDG	  PET	  scan	  
Other	  standard	  clinical	  and	  laboratory	  test	  performed	  prior	  to	  initiation	  of	  treatment	  in	  patients	  
with	  osteosarcoma	  include:	  
• History	   and	   physical	   examination,	   including	   measurement	   of	   the	   primary	   tumor	  
(circumference	  for	  limb	  tumors)	  and	  performance	  status,	  
• CBC	   with	   differential,	   comprehensive	   mineral	   panel,	   calcium,	   phosphate,	   magnesium,	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
15  
urinalysis,	  12-­‐24	  h	  urine	  collection	  for	  creatinine	  clearance,	  urine	  pregnancy	  test,	  
• Echocardiogram	   with	   quantitation	   of	   left	   ventricular	   shortening	   fraction	   prior	   to	  
administration	  of	  doxorubicin,	  and	  
• Audiogram	  to	  document	  baseline	  hearing	  prior	  to	  administration	  of	  cisplatin.	  
These	  procedures	  are	  standard	  of	  care	  for	  patients	  with	  osteosarcoma	  and	  can	  be	  performed	  
prior	  to	  informed	  consent	  and	  enrollment	  on	  this	  clinical	  trial.	  Results	  of	  these	  studies	  will	  be	  
used	   as	   baseline	   values	   and	   entered	   into	   the	   clinical	   trial	   database	   after	   patients	   have	  
consented	  and	  enrolled	  onto	  the	  trial.	  
3.1.2	   Baseline	  (Pretreatment)	  Research	  Studies	  
See	  Section	  4.1	  for	  research	  sample	  collection,	  processing,	  and	  storage	  instructions.	  
• Urinary	  AKI	  Markers	  (NAG,	  NGAL,	  KIM-­‐1)	  	  
• Plasma	  and	  urine	  magnesium	  and	  creatinine	  for	  FEMg.	  
• Serum	  cystatin	  C	  
• Serum	  bone-­‐specific	  alkaline	  phosphatase	  (BSAP)	  
• Plasma	  pre-­‐albumin	  
• Patient-­‐reported	  outcomes	  survey	  
• Upper	   arm	   (mid-­‐way	   between	   the	   shoulder	   [acromion]	   and	   elbow	   [humeral	   lateral	  
condyle])	  circumference	  with	  elbow	  extended	  and	  triceps	  skin	  fold	  thickness	  1	  cm	  above	  
site	   circumference	   was	   measured	   in	   arm	   NOT	   involved	   with	   tumor.	   These	  
measurements	   are	   performed	   to	   estimate	   nutritional	   status,	   muscle	   mass,	   and	  
creatinine	  production.	  
3.1.3	   Treatment	  with	  MAP	  Regimen	  
All	   patients	   will	   receive	   6	   cycles	   of	   the	   3-­‐drug	   MAP	   chemotherapy	   regimen	   (Methotrexate,	  
Adriamycin	   [doxorubicin]	   and	   cisPlatin)	   that	   is	   shown	   in	   Figure	   2.	   Cycles	   1	   &	   2	   will	   be	  
administered	   neoadjuvantly	   (before	   resection	   of	   the	   primary	   tumor).	   After	   recovery	   from	  
surgery,	  patients	  will	  resume	  treatment	  with	  2	  cycles	  of	  MAP	  followed	  by	  2	  cycles	  of	  MA	  (no	  
cisplatin).	  
Treatment	  arm	  specific	  treatment	  delivery	  maps	  (TDM)	  will	  be	  generated	  for	  each	  patient	  after	  
randomization	  and	  placed	  in	  the	  clinic	  chart	  to	  serve	  as	  a	  guide.	  Appendix	  1	  shows	  an	  example	  
of	  a	  TDM.	  
Standard	   treatments:	   Cycles	   1-­‐4	   of	   MAP	   are	   5	   weeks	   (35	   days)	   in	   duration.	   Cisplatin	   and	  
doxorubicin	  are	  administered	  on	  days	  1	  &	  2	  and	  HDMTX	  +	  leucovorin	  rescue	  is	  infused	  on	  days	  
22	  &	  29.	  cisplatin	  is	  not	  administered	  in	  Cycles	  5	  &	  6	  and	  the	  duration	  of	  Cycles	  5	  &	  6	  is	  4	  weeks	  
(28	   days).	   During	   cycles	   5	   &	   6,	   doxorubicin	   is	   administered	   on	   days	   1	   &	   2	   and	   HDMTX	   +	  
leucovorin	   rescue	   is	   infused	   on	   days	   15	   &	   22.	   Supportive	   care/rescue	   agents	   that	   are	  
administered	   with	   MAP	   to	   alleviate	   toxicity	   include	   antiemetics	   (see	   CHOP	   Cancer	   Center	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
16  
antiemetic	  guidelines),	  dexrazoxane	  as	  a	  cardioprotectant,	  pegfilgrastim	  to	  facilitate	  neutrophil	  
recovery	   after	   doxorubicin,	   and	   leucovorin	   as	   a	   HDMTX	   rescue	   agent.	   Intravenous	   fluid	  
hydration	   is	   administered	   with	   cisplatin	   and	   HDMTX	   to	   prevent	   nephrotoxicity,	   and	   urine	  
alkalinization	  is	  used	  to	  increase	  the	  solubility	  of	  MTX	  in	  urine.	  
Research	   Interventions:	   Pantoprazole	   will	   be	   administered	   intravenously	   with	   cisplatin	   on	   2	  
consecutive	   treatment	   cycles	   (either	   Cycles	   1	   &	   2	   or	   Cycles	   3	   &	   4),	   and	   the	   order	   of	  
pantoprazole	  administration	  will	  be	  determined	  by	  randomization	  (see	  Study	  Diagram).	  Patients	  
will	  receive	  HDMTX	  as	  4	  and	  12	  h	  infusions	  on	  2	  consecutive	  treatment	  cycles	  (Cycles	  1	  &	  2	  or	  
Cycles	  3	  &	  4),	  and	  the	  order	  of	  the	  infusion	  durations	  will	  be	  determined	  by	  randomization.	  
Drug	   doses,	   schedules,	   and	   administration	   routes:	   for	   the	   chemotherapy	   and	   supportive	  
care/rescue	  medications	  are	  listed	  below:	  
• Dexrazoxane	   375	   mg/m2/dose	   IV	   over	   15	   min	   immediately	   prior	   to	   the	   doxorubicin	  
doses	  on	  days	  1	  &	  2	  (total	  dose	  750	  mg/m2/cycle)	  of	  treatment	  cycles	  1-­‐6;	  
• DOXOrubicin	   37.5	   mg/m2/dose	   IV	   over	   15	   min	   on	   days	   1	   &	   2	   (total	   dose	   75	  
mg/m2/cycle)	  of	  treatment	  cycles	  1-­‐6	  (total	  cumulative	  dose	  450	  mg/m2);	  
• Pantoprazole	  0.3	  mg/kg	  IV	  over	  15	  min	  immediately	  prior	  to	  cisplatin	  as	  a	  loading	  dose	  
on	  days	  1	  &	  2	  followed	  by	  1.3	  mg/kg	  IV	  infused	  over	  4	  h	  concurrent	  with	  the	  4	  h	  cisplatin	  
infusion	  on	  days	  1	  &	  2	  of	  treatment	  Cycles	  1	  &	  2	  (Treatment	  Arms	  1,	  3)	  OR	  Cycles	  3	  &	  4	  
(Treatment	  Arms	  2,	  4);	  
• CISplatin	  60	  mg/m2/dose	  infused	  IV	  over	  4	  h	  on	  days	  1	  &	  2	  (total	  dose	  120	  mg/m2/cycle)	  
of	  treatment	  cycles	  1-­‐4;	  
• Pegfilgrastim	   0.1	   mg/kg	   (6	   mg	   for	   patients	   weighing	   ≥45	   kg)	   SC	   on	   day	   3	   or	   4	   of	  
treatment	  cycles	  1-­‐6;	  
• Methotrexate	  12	  g/m2	  (maximum	  dose	  20	  g)	  infused	  IV	  over	  4	  OR	  12	  h	  on	  days	  22	  &	  29	  
of	  Cycles	  1-­‐4	  and	  on	  days	  15	  and	  22	  of	  Cycles	  5	  &	  6	  (HDMTX	  infusion	  duration	  on	  Cycles	  
5	  &	  6	  based	  on	  patient	  tolerance	  on	  cycles	  1-­‐4);	  
• Leucovorin	  15	  mg/m2	  IV	  or	  PO	  every	  6	  h	  until	  serum	  MTX	  <0.1	  mcM	  on	  days	  23	  and	  30	  
of	   cycles	   1-­‐4	   and	   days	   16	   &	   23	   of	   cycles	   5	   &	   6,	   starting	   24	   h	   after	   the	   start	   of	   the	  
HDMTX	  infusion.	  
Intravenous	   fluid	   hydration	   guidelines:	   Intravenous	   fluid	   hydration	   should	   be	   administered	  
prior	  to,	  during	  and	  after	  cisplatin	  and	  HDMTX.	  In	  addition,	  the	  urine	  should	  be	  alkalinized	  to	  a	  
pH	  of	  ≥7	  for	  HDMTX	  until	  serum	  MTX	  <1	  mcM	  by	  including	  sodium	  bicarbonate	  or	  equivalent	  
base	  in	  the	  IV	  fluids.	  
Cisplatin:	  
• Prior	  to	  the	  first	  dose	  of	  cisplatin	  on	  day	  1	  of	  the	  cycle,	  infuse	  a	  normal	  saline	  (0.9%	  
NaCl)	  fluid	  bolus	  of	  750	  mL/m2	  over	  1	  h.	  
• Cisplatin	  is	  diluted	  in	  0.9%	  NaCl	  to	  a	  final	  cisplatin	  concentration	  of	  100	  mg/L.	  Therefore,	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
17  
patients	  will	  receive	  150	  mL/m2/h	  of	  this	  solution	  over	  the	  4	  hour	  drug	  infusion.	  
• After	  the	  cisplatin	  infusion	  is	  completed,	  infuse	  100	  mL/m2/h	  D5	  0.45%	  NaCl	  +	  20	  mEq/L	  
KCl	  until	  at	  least	  12	  h	  after	  the	  second	  cisplatin	  infusion	  is	  completed	  and	  the	  patient	  is	  
tolerating	  PO	  fluids.	  
• Monitor	  Input	  and	  Output,	  at	  3hr	  or	  6hr	  after	  start	  of	  cisplatin	  	  
o If	  urine	  Output	  >	  total	  Input	  by	  100	  mL/m2/hr	  over	  the	  previous	  3	  hr,	  consider	  
0.9%	  NaCl	  bolus	  (10-­‐15	  cc/kg)	  
o If	  total	  Input	  >	  urine	  Output	  by	  100	  mL/m2/hr	  over	  the	  previous	  3	  hr,	  consider	  
furosemide	  (0.5	  mg/kg,	  MAX	  dose	  20	  mg)	  to	  promote	  urine	  output.104,105	  
HDMTX:	  
• Prior	  to	  the	  start	  of	  a	  HDMTX	  infusion,	  infuse	  D5	  0.22%	  NaCl	  with	  40	  mEq/L	  NaHCO3	  
(sodium	  biocarbonate)	  or	  equivalent	  base	  as	  a	  fluid	  bolus	  of	  750	  mL/m2	  over	  1	  h.	  
• Continue	  hydration	  with	  D5	  0.22%	  NaCl	  with	  40	  mEq/L	  NaHCO3	  (sodium	  bicarbonate)	  or	  
equivalent	  base	  at	  100	  mL/m2/h	  until	  serum	  MTX	  <1	  mcM	  and	  PO	  fluids	  are	  tolerated.	  
Acetate	  can	  be	  substituted	  if	  bicarbonate	  is	  not	  available	  at	  a	  1:1	  dose	  conversion.	  
• If	   urine	   pH	   <7	   administer	   0.5	   mEq/kg	   NaHCO3	   IV.	   Recheck	   urine	   pH	   ≥30	   min	   after	  
NaHCO3	   bolus	   is	   complete.	   Repeat	   NaHCO3	   bolus	   until	   urine	   pH	   ≥7	   and	   consider	  
increasing	  dose	  of	  bicarbonate	  in	  maintenance	  fluids	  if	  urine	  pH	  consistently	  <7.	  
• Once	   urine	   pH	   ≥7	   start	   HDMTX	   infusion.	  
Continue	  to	  check	  urine	  pH	  every	  8	  hours	  
and	   administer	   0.5	   mEq/kg	   NHCO3	   IV	   if	  
urine	  pH	  <7,	  until	  serum	  MTX	  <1	  mcM.	  
• HDMTX	   solution	   (25	   mg/mL)	   is	   piggy-­‐
backed	   into	   the	   hydration	   fluids	   and	  
infused	  over	  4	  OR	  12	  h	  depending	  on	  the	  
treatment	   arm	   and	   cycle	   (see	   Study	  
Diagram).	  
Leucovorin	   dosing	   guidelines	   including	   guide-­‐
lines	  for	  delayed	  MTX	  excretion:	  	  
• The	   dose	   and	   duration	   of	   leucovorin	   rescue	  
are	  
determined	  
by	  
the	  
plasma	  
MTX	  
concentration.	  Leucovorin	  rescue	  should	  start	  
no	   later	   than	   24	   h	   after	   the	   start	   of	   the	  
HDMTX	   infusion	   and	   continue	   until	   the	  
plasma	  MTX	  concentration	  is	  <0.1	  mcM.	  
• Leucovorin	   15	   mg/m2	   IV	   or	   PO	   every	   6	   h	  
starting	   24	   h	   after	   the	   start	   of	   the	   HDMTX	  
	  
Figure	  11:	  	  Concentration	  time	  curves	  for	  MTX	  
(12	  g/m2)	  infusions	  over	  4	  and	  12	  h	  
generated	  by	  fitting	  a	  2-­‐compartment	  
pharmacokinetic	  model	  to	  the	  mean	  
plasma	  MTX	  concentrations	  from	  
1,045	  infusions	  of	  12	  g/m2	  over	  4	  h.5	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
18  
infusion	  on	  days	  23	  and	  30	  of	  cycles	  1-­‐4	  and	  days	  16	  &	  
23	   of	   cycles	   5	   &	   6	   and	   administered	   until	   serum	   MTX	  
<0.1	  mcM.	  
• Serum	  MTX	  levels	  will	  vary	  based	  on	  the	  duration	  of	  the	  
HDMTX	  infusion	  (Figure	  11).	  Table	  3	  lists	  the	  expected	  
plasma	   MTX	   concentrations	   at	   various	   times	   after	   a	   4	  
and	  12	  h	  infusion.	  Please	   contact	   the	   PI	   or	   study	   Co-­‐
investigators	   at	   any	   time	   for	   questions	   or	   concerns	  
about	  MTX	  levels.	  
• If	  a	  patient	  experiences	  MTX	  nephrotoxicity	  and	  delayed	  
MTX	   clearance,	   the	   leucovorin	   dose	   or	   schedule	   may	  
need	  adjustment	  based	  on	  plasma	  MTX	  concentration,	  
or	  the	  patient	  may	  require	  glucarpidase	  to	  lower	  plasma	  MTX	  concentrations.	  The	  study	  PI	  
or	  a	  co-­‐Investigator	  should	  be	  contacted	  immediately	  if	  the	  plasma	  MTX	  concentration	  is	  
>100	  mcM	  at	  hour	  24,	  >50	  mcM	  at	  hour	  28-­‐40,	  or	  >10	  mcM	  at	  hour	  48	  or	  later.	  Leucovorin	  
should	  be	  escalated	  to	  15	  mg/m2	  IV	  or	  PO	  every	  3	  h	  if	  the	  plasma	  MTX	  concentration	  is	  
>2-­‐fold	   higher	   than	   expected	   (Table	   3)	   AND	   >1	   mcM.	   The	   every	   6	   h	   schedule	   can	   be	  
resumed	  when	  the	  plasma	  MTX	  concentration	  is	  <1	  mcM.	  
3.1.4	   Criteria	  for	  Starting	  Treatment	  
CISplatin/DOXOrubicin	  (Cycle	  1-­‐4)	  or	  DOXOrubicin	  alone	  (Cycle	  5&6):	  
Delay	  treatment	  until	  the	  following	  conditions	  are	  met:	  
• ANC	  >750/mcL	  and	  platelet	  count	  >75,000/mcL	  
• Oral	  mucositis	  grade	  <2	  
• Hyperbilirubinemia	  (total)	  grade	  <3	  and	  ALT/AST	  elevation	  grade	  <4	  
• Normal	   serum	   creatinine	   for	   age	   and	   gender	   (see	   Table	   in	   eligibility	   criteria)	   OR	  
creatinine	  clearance	  >70	  mL/min/1.73	  m2	  (Cycles	  1-­‐4	  only).	  If	  renal	  function	  does	  not	  
recover	  after	  a	  one	  week	  delay,	  administer	  doxorubicin	  alone.	  
• Adequate	   wound	   healing	   (determined	   by	   the	   surgeon)	   after	   resection	   of	   the	   primary	  
tumor	  (prior	  to	  Cycle	  3)	  or	  after	  resection	  of	  pulmonary	  metastases.	  
HDMTX:	  
Delay	  treatment	  until	  the	  following	  conditions	  are	  met:	  
• ANC	  >250/mcL	  and	  platelet	  count	  >50,000/mcL	  
• Oral	  mucositis	  grade	  <2	  
• Hyperbilirubinemia	  grade	  <3	  and	  ALT/AST	  elevation	  grade	  <4	  
• Normal	  serum	  creatinine	  for	  age	  &	  gender	  OR	  creatinine	  clearance	  >70	  mL/min/1.73	  m2	  
Table	  3.	  Expected	  MTX	  levels	  by	  
infusion	  duration.	  (EOI,	  end	  
of	  infusion)	  
	  
Plasma	  MTX	  [mcM]	  
Sample	  
time	  (h)	  
4	  h	  
Infusion	  
12	  h	  
Infusion	  
EOI	  
1200	  
600	  
24	  
15	  
40	  
36	  
3	  
5	  
48	  
1	  
2	  
60	  
0.5	  
1	  
72	  
0.3	  
0.4	  
96	  
<0.1	  
<0.1	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
19  
3.1.5	   Dose	  Modifications	  
Modification	  of	  the	  doses	  of	  cisplatin,	  doxorubicin,	  and	  HDMTX	  is	  required	  if	  toxicity	  for	  the	  
drugs	  exceeds	  the	  thresholds	  listed	  in	  the	  Tables	  below	  on	  a	  prior	  treatment	  cycle.	  The	  percent	  
of	   full	   protocol	   prescribed	   dose	   should	   be	   recorded	   on	   the	   TDM	   in	   the	   column	   labeled	   “%	  
Dose”.	  If	  the	  dose	  is	  reduced	  by	  25%,	  then	  the	  %	  Dose	  should	  be	  recorded	  as	  75%	  (75%	  of	  the	  
full	  dose).	  
Cisplatin:	  
Toxicity	  
Severity	  
Modification	  
Ototoxicity	  
>30	  dB	  threshold	  at	  2	  kHz	  
Discontinue	  cisplatin	  
Nephrotoxicity	  
Serum	  creatinine	  >ULN	  for	  age	  and	  
gender	  OR	  creatinine	  clearance	  <70	  
mL/min/1.73	  m2	  
Hold	  cisplatin	  until	  criterion	  in	  
Section	  3.1.4	  is	  met	  
Neuropathy*	  
Grade	  >2	  motor	  or	  sensory	  
neuropathy	  
Hold	  cisplatin	  until	  resolved	  to	  grade	  
≤2	  
*	  
See	  Appendix	  2	  for	  toxicity	  grading	  scale	  
Doxorubicin:	  
Toxicity	  
Severity	  
Modification	  
Myelosuppression	   Planned	  day	  22	  HDMTX	  is	  delayed	  
by	  >10d	  (cycle	  day	  >32)	  due	  to	  ANC	  
<250/mcL	  or	  platelet	  count	  
<50,000/mcL	  
Reduce	  doxorubicin	  dose	  by	  20%	  
Cardiotoxicity	  
SF	  <27%	  (confirmed	  with	  repeat	  
Echocadiogram	  1	  week	  later)	  
Hold	  doxorubicin	  until	  recovery	  to	  
SF	  >28%	  
Mucositis	  
Grade	  4	  related	  to	  doxorubicin	  
Reduce	  doxorubicin	  dose	  by	  20%	  
HDMTX:	  
Toxicity	  
Severity	  
Modification	  
Nephrotoxicity	  
Serum	   creatinine	   >ULN	   for	   age	   and	  
gender	   OR	   creatinine	   clearance	   <70	  
mL/min/1.73	  m2	  
Hold	  HDMTX.	  Omit	  dose	  if	  renal	  
function	  has	  not	  recovered	  after	  a	  7-­‐
day	  delay	  
Mucositis	  
Grade	  4	  related	  to	  HDMTX	  
Reduce	  HDMTX	  by	  50%	  
3.1.6	   Local	  Control	  of	  Primary	  Tumor	  
Complete	  resection	  of	  the	  primary	  tumor	  is	  the	  optimal	  form	  of	  local	  therapy	  and	  is	  performed	  
after	   two	   5-­‐week	   cycles	   of	   neoadjuvant	   MAP	   chemotherapy	   (11	   weeks	   after	   the	   start	   of	  
therapy).	  A	  limb	  salvage	  procedure	  is	  indicated	  if	  the	  neurovascular	  bundle	  is	  not	  extensively	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
20  
involved,	  the	  tumor	  can	  be	  resected	  en	  bloc	  with	  tumor-­‐free	  margins,	  and	  reconstruction	  with	  
an	  endoprosthesis	  to	  replace	  the	  involved	  bone	  and	  joint	  is	  feasible.	  If	  a	  limb	  salvage	  procedure	  
is	  not	  indicated,	  an	  amputation	  of	  the	  involved	  portion	  of	  the	  limb	  will	  be	  performed.	  
Radiation	  therapy	  is	  reserved	  for	  patients	  who	  have	  unresectable	  primary	  tumors	  (e.g.,	  spine	  
tumors).	  The	  timing	  of	  radiation	  therapy	  will	  be	  based	  on	  symptoms,	  such	  as	  pain,	  response	  to	  
neoadjuvant	   chemotherapy,	   and	   ability	   to	   deliver	   full	   dose	   intensity	   of	   the	   chemotherapy	  
regimen.	   Radiation	   is	   less	   effective	   than	   surgery	   but	   can	   achieve	   local	   control	   in	   some	  
patients.106,107	  
3.1.7	   Management	  of	  Metastatic	  Tumors	  
Complete	  resection	  of	  overt	  pulmonary	  metastases	  that	  are	  present	  at	  diagnosis	  can	  enhance	  
survival	  chances.11,108	  Median	  sternotomy	  or	  bilateral	  thoracotomies	  with	  manual	  palpation	  of	  
both	  lungs	  is	  recommended	  even	  in	  patients	  with	  unilateral	  pulmonary	  metastases	  on	  CT	  scan.	  
Timing	  of	  resection	  of	  metastatic	  disease	  is	  left	  to	  the	  clinical	  judgment	  of	  the	  oncologist	  and	  
surgeons.	  
3.2	   Randomization	  
Patients	   will	   be	   randomized	   to	   one	   of	   4	   treatment	   arms	   (see	   Study	   Diagram),	   which	   will	  
determine	  the	  order	  in	  which	  they	  receive	  pantoprazole	  with	  cisplatin	  (Cycles	  1&2	  vs.	  Cycles	  
3&4)	  and	  the	  order	  in	  which	  they	  will	  receive	  HDMTX	  as	  a	  4	  or	  12	  h	  infusion	  (Cycles	  1&2	  vs.	  
Cycles	  3&4).	  A	  block	  randomization	  procedure	  will	  be	  used	  so	  that	  3	  of	  the	  first	  12	  subjects	  will	  
be	  randomly	  assigned	  to	  each	  of	  the	  4	  treatment	  arms	  (three	  patient	  per	  arm)	  and	  3	  of	  subjects	  
13	  to	  24	  will	  be	  randomly	  assigned	  to	  each	  of	  the	  4	  arms	  (six	  patients	  per	  arm	  when	  enrollment	  
is	  completed).	  Subjects	  who	  are	  unable	  to	  complete	  4	  cycles	  of	  therapy	  may	  be	  replaced,	  and	  
the	  new	  subject	  will	  be	  placed	  on	  the	  same	  treatment	  arm	  as	  the	  subject	  being	  replaced.	  
3.3	   Study	  Duration,	  Enrollment	  and	  Number	  of	  Sites	  
3.3.1	   Duration	  of	  Study	  
The	   duration	   of	   study	   participation	   will	   be	   approximately	   8	   months.	   The	   screening	   process	  
including	   biopsy	   of	   the	   primary	   tumor	   for	   diagnosis	   will	   take	   up	   to	   2	   weeks.	   The	   first	   4	  
treatment	  cycles	  are	  5	  weeks	  each	  (total,	  20	  weeks)	  and	  cycles	  5	  and	  6	  are	  4	  weeks	  each	  (total,	  
8	   weeks).	   The	   time	   for	   recovery	   from	   surgery	   to	   resect	   the	   primary	   tumor	   after	   Cycle	   2	   is	  
usually	   1-­‐2	   weeks.	   Endpoints	   for	   this	   trial	   are	   measured	   during	   therapy	   and	   at	   the	   end	   of	  
therapy	  visit	  after	  the	  last	  HDMTX	  infusion.	  
3.3.2	   Number	  of	  Subjects	  and	  Number	  of	  Study	  Sites	  
This	  pilot	  study	  will	  be	  conducted	  at	  CHOP.	  We	  plan	  to	  study	  24	  subjects.	  Patients	  who	  do	  not	  
complete	   4	   cycles	   of	   treatment	   for	   any	   reason	   may	   be	   replaced.	   We	   may	   enroll	   up	   to	   28	  
subjects	  in	  order	  to	  have	  24	  fully	  evaluable	  subjects.	  Based	  on	  the	  number	  of	  children	  with	  OS	  
seen	  at	  CHOP	  annually,	  we	  expect	  accrual	  to	  be	  complete	  in	  2	  to	  3	  years.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
21  
3.4	   Study	  Population	  
3.4.1	   Study	  Enrollment/Registration	  
Subjects	  are	  enrolled	  on	  study	  after	  fulfilling	  the	  eligibility	  criteria	  listed	  below.	  Treatment	  arm	  
will	  be	  assigned	  by	  the	  Study	  PI	  based	  on	  a	  spreadsheet	  provided	  by	  the	  Study	  Biostatistician.	  
Demographic	  information,	  results	  of	  screening	  and	  baseline	  studies,	  tumor	  site(s)	  and	  histology,	  
and	   treatment	   arm	   will	   be	   entered	   into	   OnCore	   CRM.	   Patients	   must	   be	   enrolled	   and	  
randomized	  prior	  to	  starting	  neoadjuvant	  chemotherapy.	  
3.4.2	   Inclusion	  Criteria	  
• Age:	  <30	  years	  of	  age	  
• Diagnosis:	  histological	  diagnosis	  of	  high-­‐grade	  osteosarcoma	  (Section	  3.1.1)	  
• Site:	   Extremity	   or	   central	   axis	   (including	   craniofacial)	   primary	   tumor;	   localized	   or	  
metastatic	  
• Prior	  therapy:	  No	  prior	  chemotherapy	  or	  radiation	  therapy	  for	  osteosarcoma.	  Subjects	  
who	  develop	  osteosarcoma	  as	  a	  second	  cancer	  are	  eligible	  if	  they	  have	  not	  previously	  
received	  cisplatin,	  doxorubicin	  or	  other	  anthracyclines,	  or	  MTX	  
• Kidney	  function:	  Serum	  creatinine	  at	  or	  below	  the	  upper	  limit	  of	  normal	  (ULN)	  for	  age	  
and	  gender:	  
Age	  
Maximum	  Serum	  
Creatinine	  (mg/dL)	  
	  
Male	  
Female	  
1	  mo	  to	  <	  6	  months	  
0.4	  
0.4	  
6	  mo	  to	  <	  1	  year	  
0.5	  
0.5	  
1	  year	  to	  	  <	  2	  years	  
0.6	  
0.6	  
2	  to	  <	  6	  years	  
0.8	  
0.8	  
6	  to	  <	  10	  years	  
1	  
1	  
10	  to	  <	  13	  years	  
1.2	  
1.2	  
13	  to	  <	  16	  years	  
1.5	  
1.4	  
≥	  16	  years	  
1.7	  
1.4	  
Derived	  for	  Schwartz	  et	  a.	  J	  Peds	  106:522,	  1985	  utilizing	  
child	  length	  and	  stature	  data	  published	  by	  the	  CDC	  	  
• Cardiac	  function:	  Shortening	  fraction	  on	  echocardiogram	  >28%	  
• Hearing:	  Hearing	  level	  threshold	  ≤25	  dB	  at	  all	  frequencies	  in	  both	  ears	  to	  be	  evaluable	  
for	  evaluation	  of	  pantoprazole’s	  effect	  on	  cisplatin	  ototoxicity.	  Patients	  with	  hearing	  loss	  
can	  be	  enrolled	  but	  will	  not	  be	  evaluable	  for	  ototoxicity	  objective.	  
• Hematological	   function:	   Absolute	   neutrophil	   count	   >1,000/mcL	   and	   platelet	   count	  
>100,000/mcL	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
22  
3.4.3	   Exclusion	  Criteria	  
• Receiving	  H2	  antagonists	  (cimetidine,	  ranitidine,	  famotidine,	  nizatidine)	  or	  proton	  pump	  
inhibitors	   (lansoprazole,	   omeprazole,	   pantoprazole,	   esomeprazole,	   raberprazole,	  
dexlansoprazole)	   AND	   unable	   to	   hold	   the	   drug	   for	   24	   h	   prior	   to	   and	   24	   h	   after	   each	  
cisplatin	  course	  on	  cycles	  1-­‐4.	  
• Inability	  to	  tolerate	  a	  PPI	  
• Pregnant	  or	  breastfeeding	  
• Unable	  to	  cooperate	  with	  research	  procedures	  
4.	   RESEARCH	  PROCEDURES	  
Refer	   to	   Treatment	   arm	   specific	   TDMs	   for	   schedule	   of	   drug	   administration	   and	   Schedule	   of	  
Study	  Procedures	  for	  timing	  of	  standard	  of	  care	  and	  research	  procedures.	  
4.1	   Research	  Studies	  during	  Treatment	  
See	  Schedule	  of	  Study	  Procedures	  table	  for	  timing	  of	  standard	  of	  care	  and	  research	  procedures.	  
Test	  
Specimen	  
Tube	  
Volume	  
Collection	  
Processing	  
Storage	  
Lab/Assay	  
KIM-­‐1	  
Spot	  urine	  
Urine	  
container	  
>5	  mL	  
Prior	  to	  cisplatin	  
D1,	  EOI	  D2,	  D7;	  
prior	  to	  each	  
HDMTX	  &	  24	  
after	  the	  start	  of	  
infusion;	  pre-­‐op;	  
C5D1	  
Centrifuge	  
-­‐70°C	  
CTSU	  Core	  
NGAL	  
NAG	  
Cystatin	  C	  
Serum	  
Gold	  top	  
SST-­‐clot	  
activator	  
3	  mL	  
Prior	  to	  cisplatin	  
and	  HDMTX;	  D7;	  
pre-­‐op;	  C5D1;	  
post-­‐inf	  IF	  sCr	  
≥50%	  above	  
baseline	  	  
Centrifuge	  
-­‐70°C	  
UPenn	  Core	  
Mg,	  Cr	  
Plasma	  
Light	  
green	  top	  
0.5	  mL	  
Prior	  to	  cisplatin	  
D1,	  D2	  &	  D7;	  
C5D1	  
Send	  to	  Clinical	  
Lab	  
Room	  
temp	  
Clinical	  Lab	  
Mg,	  Cr	  
Spot	  urine	  
Urine	  
container	  
>2	  mL	  
Prior	  to	  cisplatin	  
D1,	  D2	  &	  D7;	  
C5D1	  
Send	  to	  Clinical	  
Lab	  
Room	  
temp	  
Clinical	  Lab	  
BSAP	  
Serum	  
Red	  top	  
2	  mL	  
Baseline,	  pre-­‐
op,	  C3D1	  &	  EOT	  
Clot,	  centrifuge,	  
separate	  serum	  
-­‐70°	  C	  
CTSU	  Core	  
Pre-­‐albumin	  
Plasma	  
Light	  
green	  top	  
1	  mL	  
Baseline,	  D1	  C2-­‐
6,	  Pre-­‐op,	  EOT	  
Send	  to	  Clinical	  
Lab	  
Room	  
temp	  
Clinical	  Lab	  
MTX	  
Urine	  
Conical	  
tube	  	  
1	  mL	  
EOI	  of	  each	  
HDMTX	  C1-­‐4	  
Pipette	  1	  mL	  
urine	  into	  
conical	  tube	  w/	  
9	  mL	  buffer	  
-­‐70°	  C	  
PK	  Core	  
HPLC/MS/MS	  
Abbreviations:	  EOI,	  end	  of	  infusion;	  Mg,	  magnesium;	  Cr,	  creatinine;	  BSAP,	  bone-­‐specific	  alkaline	  phosphatase;	  inf,	  
infusion;	  EOT,	  end	  of	  therapy;	  MTX,	  methotrexate	  concentration	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
23  
4.2	   Post-­‐Therapy	  Follow-­‐up	  
Periodic	   follow-­‐up	   and	   restaging	   is	   standard	   of	   care	   for	   patients	   who	   have	   completed	  
treatment.	  Guidelines	  for	  following	  patients	  after	  completion	  of	  treatment	  are	  provided	  in	  the	  
table	  below:	  
	  
Months	  Post-­‐Treatment	  
Procedure	  
3	  
6	  
9	  
12	  
15	  
18	  
21	  
24	  
30	  
36	  
42	  
48	  
54	  
60	  
History,	  physical,	  PS	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
CBC,	  CMP,	  Mg,	  PO4	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
	  
	  
	  
	  
	  
	  
Chest	  CT	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
X	  
	  
	  
	  
	  
	  
	  
MRI	  of	  primary1	  
X	  
X	  
	  
X	  
	  
X	  
	  
X	  
	  
	  
	  
	  
	  
	  
Chest	  X-­‐ray	  
	  
	  
	  
	  
	  
	  
	  
	  
X	  
X	  
X	  
X	  
X	  
X	  
X-­‐ray	  of	  primary	  
	  
	  
	  
	  
	  
	  
	  
	  
X	  
X	  
X	  
X	  
X	  
X	  
Bone	  or	  PET	  scan2	  
	  
X	  
	  
X	  
	  
X	  
	  
X	  
	  
	  
	  
	  
	  
	  
Audiogram	  
	  
	  
	  
X	  
	  
	  
	  
X	  
	  
	  
	  
	  
	  
X	  
Echocardiogram	  
	  
	  
	  
X	  
	  
	  
	  
X	  
	  
X	  
	  
X	  
	  
X	  
BSAP3	  
Draw	  sample	  if	  subject	  has	  evidence	  of	  tumor	  recurrence	  
Arm	  circum/Skin	  fold3	  
X	  
X	  
	  
X	  
	  
	  
	  
X	  
	  
X	  
	  
X	  
	  
X	  
1	  
Substitute	  CT	  scan	  if	  metal	  in	  endoprothesis	  does	  not	  allow	  for	  MRI	  to	  be	  performed	  
2	  
Only	  if	  positive	  for	  metastatic	  disease	  at	  baseline	  
3	  
Research	  procedure	  
4.3	   Concomitant	  Medications	  
H2-­‐antagonists	   (cimetidine,	   ranitidine,	   famotidine,	   nizatidine)	   and	   proton	   pump	   inhibitors	  
(lansoprazole,	  omeprazole,	  pantoprazole,	  esomeprazole,	  raberprazole,	  dexlansoprazole)	  should	  
NOT	   be	   administered	   for	   24	   h	   prior	   to	   and	   24	   h	   after	   cisplatin	   on	   Cycles	   1-­‐4.	   Patients	   will	  
receive	  pantoprazole	  with	  cisplatin	  on	  Cycles	  1&2	  OR	  Cycles	  3&4.	  
Probenecid,	  NSAIDS,	  sulfonamides	  (including	  co-­‐trimoxazole),	  and	  penicillins	  should	  NOT	  be	  
administered	  concurrently	  with	  HDMTX.	  These	  drugs	  can	  interfere	  with	  the	  secretion	  of	  MTX	  by	  
the	  renal	  tubule.	  
Patients	  should	  receive	  co-­‐trimoxazole	  2.5	  mg/kg/dose	  twice	  daily	  on	  2	  consecutive	  days	  per	  
week	   to	   prevent	   pneumocystis	   pneumonia.	   Co-­‐trimoxazole	   should	   NOT	   be	   administered	  
concurrently	  with	  HDMTX.	  
Antiemetics	   should	   be	   administered	   prior	   to	   and	   as	   needed	   during	   and	   after	   chemotherapy	  
according	  to	  the	  CHOP	  Antiemetic	  guidelines	  available	  on	  the	  Cancer	  Center	  Website.	  
4.4	   Subject	  Completion/Withdrawal	  
Subjects	  will	  be	  followed	  (Section	  4.2)	  until:	  
• Five	  years	  after	  completing	  treatment	  
• Relapse	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
24  
• Death	  from	  any	  cause	  
Subjects	   may	   voluntarily	   withdraw	   from	   this	   study	   at	   any	   time	   but	   should	   complete	   the	  
standard	  of	  care	  chemotherapy	  regimen	  and	  resection	  of	  their	  primary	  and	  metastatic	  tumor	  as	  
described	  in	  this	  protocol.	  
5.	   STUDY	  ENDPOINTS	  AND	  EVALUATIONS	  
The	  timing	  of	  study	  endpoints,	  as	  well	  as	  standard	  of	  care	  procedures,	  is	  shown	  in	  the	  Schedule	  
of	  Study	  Procedures.	  
5.1	   Primary	  Endpoints	  
• Urinary	   biomarkers	   of	   acute	   kidney	   injury	   (KIM-­‐1,	   NAG,	   NGAL)	   after	   each	   course	   of	  
cisplatin	   (days	   2	   and	   7)	   and	   each	   HDMTX	   infusion	   (24	   h	   and	   day	   7)	   on	   cycles	   1-­‐4	  
compared	  to	  baseline	  (pre-­‐infusion)	  values	  
• Serum	   cystatin-­‐C	   and	   creatinine	   prior	   to	   each	   course	   of	   cisplatin	   and	   each	   HDMTX	  
infusion	   on	   cycles	   1-­‐4	   and	   post-­‐infusion	   cystatin-­‐C	   and	   BMP	   if	   serum	   creatinine	  
increases	  by	  ≥50%	  compared	  to	  pre-­‐infusion	  baseline	  value	  
• FEMg	  (simultaneous	  serum	  and	  spot	  urine	  for	  Mg	  and	  creatinine)	  prior	  to	  the	  start	  of	  
the	  cisplatin	  infusions	  on	  days	  1&2	  and	  day	  7	  of	  cycles	  1-­‐4	  
• Plasma	  and	  urine	  MTX	  concentrations	  at	  the	  end	  of	  each	  HDMTX	  infusion	  on	  cycles	  1-­‐4	  
• Audiograms	  prior	  to	  cycles	  1-­‐4	  and	  at	  the	  end	  of	  therapy.	  
5.2	   Secondary	  Endpoints	  
• The	  incidence	  and	  severity	  (CTCAE	  v.4	  grade)	  of	  safety	  events	  listed	  in	  Section	  5.4,	  
• Response	   of	   the	   primary	   tumor	   to	   the	   first	   2	   treatment	   cycles	   will	   be	   assessed	   by	  
quantifying	   the	   change	   in	   tumor	   volume	   on	   MRI	   after	   treatment	   relative	   to	   the	   pre-­‐
treatment	   tumor	   volume	   using	   the	   log	   ratio	   method	   [log(Vt/V0),	   where	   Vt	   is	   tumor	  
volume	   post-­‐treatment	   and	   V0	   is	   tumor	   volume	   at	   baseline]109	   and	   quantifying	   the	  
percent	  tumor	  necrosis	  in	  the	  resected	  tumor	  specimens	  histologically,	  
• The	  new	  biomarkers	  of	  AKI	  (KIM-­‐1,	  NAG,	  NGAL)	  and	  GFR	  (cystatin	  C)	  will	  be	  correlated	  
with	  the	  current	  standard	  clinical	  laboratory	  methods	  of	  assessing	  renal	  function	  with	  
serum	  creatinine,	  estimated	  creatinine	  clearance,	  urinalysis	  and	  FEMg,	  
• A	  tissue	  microarray	  will	  be	  constructed	  from	  the	  paraffin	  blocks	  containing	  the	  initial	  
biopsy	   specimen,	   the	   resected	   primary	   tumor	   and	   resected	   metastatic	   lesion	   (if	  
available)	  in	  the	  Pathology	  Core	  Laboratory.	  A	  slide	  cut	  from	  the	  top	  of	  the	  block	  will	  be	  
used	   to	   identify	   areas	   of	   viable	   tumor,	   which	   will	   then	   be	   marked	   on	   the	   block.	   IHC	  
staining	  will	  be	  performed	  for	  proteins	  responsible	  for	  resistance	  to	  MAP	  agents	  and	  for	  
protein	  targets	  of	  new	  agents	  under	  development,	  
• Serum	  bone	  specific	  alkaline	  phosphatase	  will	  be	  measured	  at	  baseline,	  pre-­‐	  and	  post-­‐
op,	  end	  of	  treatment	  and	  at	  the	  time	  of	  relapse	  and	  correlated	  with	  tumor	  volume,	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
25  
• An	  osteosarcoma-­‐specific	  patient	  reported	  outcome	  survey	  (Appendix	  3)	  developed	  for	  
this	  study	  will	  be	  administered	  prior	  to	  each	  treatment	  cycle,	  prior	  to	  surgery	  and	  at	  the	  
end	  of	  therapy,	  
• Weight,	  arm	  circumference	  (muscle	  mass),	  skin	  fold	  thickness	  (body	  fat),	  pre-­‐albumin,	  
and	   use	   of	   supplemental	   feedings	   via	   NG	   tube	   or	   G-­‐tube	   will	   be	   used	   to	   assess	   the	  
impact	  of	  treatment	  on	  nutritional	  status	  over	  the	  course	  of	  therapy.	  
5.3	   Efficacy	  Evaluations	  
• The	  effect	  of	  prolonging	  the	  duration	  of	  the	  HDMTX	  infusion	  to	  12	  h	  will	  be	  assessed	  by	  
comparing	   urinary	   biomarkers	   of	   AKI	   and	   serum	   cystatin	   C	   and	   creatinine	   measured	  
prior	  to,	  24	  h	  and	  7	  days	  after	  the	  start	  of	  each	  HDMTX	  infusion	  (n=8).	  All	  subjects	  will	  
receive	  four	  HDMTX	  infusion	  durations	  of	  4	  h	  and	  four	  HDMTX	  infusion	  durations	  of	  12	  
h	  in	  this	  crossover	  design,	  and	  subjects	  will	  serve	  as	  their	  controls.	  Measurement	  of	  the	  
urine	  MTX	  concentration	  at	  the	  end	  of	  the	  infusion	  will	  be	  used	  to	  determine	  whether	  
urinary	  MTX	  concentrations	  exceed	  the	  solubility	  limit	  for	  each	  HDMTX	  infusion.	  
• The	  effect	  of	  IV	  pantoprazole	  on	  cisplatin	  nephrotoxicity	  will	  be	  assessed	  by	  comparing	  
urinary	  biomarkers	  of	  AKI,	  FEMg	  and	  serum	  cystatin	  C	  and	  creatinine	  measured	  prior	  to,	  
at	  the	  end	  of	  the	  day	  2	  infusion,	  and	  on	  day	  7	  each	  cisplatin	  course	  (n=4).	  All	  subjects	  
will	   receive	   cisplatin	   with	   and	   without	   pantoprazole	   in	   this	   crossover	   design,	   and	  
subjects	  will,	  therefore,	  serve	  as	  their	  controls.	  
• The	   effect	   of	   IV	   pantoprazole	   on	   cisplatin	   ototoxicity	   will	   be	   assessed	   by	   comparing	  
audiograms	   performed	   prior	   to	   each	   cisplatin	   infusion	   and	   at	   the	   end	   of	   therapy.	   All	  
subjects	  will	  receive	  cisplatin	  with	  and	  without	  pantoprazole	  in	  this	  crossover	  design,	  
and	  subjects	  will,	  therefore,	  serve	  as	  their	  own	  controls.	  
• Event-­‐free	  and	  overall	  survival	  will	  be	  monitored	  but	  are	  not	  scientific	  objectives	  on	  this	  
trial.	  An	  event	  is	  defined	  as	  tumor	  progression,	  relapse	  or	  death	  from	  any	  cause.	  
5.4	   Safety	  Evaluations	  
• More	  than	  90%	  of	  patients	  receiving	  MAP	  chemotherapy	  will	  experience	  severe	  (CTCAE	  
grade	  3	  or	  4)	  acute	  toxicity.	  The	  most	  common	  acute	  toxicities	  are	  nausea	  and	  vomiting,	  
neutropenia,	   thrombocytopenia,	   fever/infection,	   oral	   mucositis,	   and	   hepatotoxicity	  
(elevated	   serum	   transaminases	   and	   bilirubin).14,16,23	   Neutropenic	   fever	   or	   infection	  
necessitates	  hospital	  admission	  and	  empirical	  antibiotics.	  
• The	  following	  adverse	  events	  will	  be	  tracked	  by	  treatment	  cycle:	  
o Nadir	  neutrophil	  count	  and	  duration	  of	  severe	  (ANC	  <500/mcL)	  neutropenia	  
o Nadir	  platelet	  count	  and	  number	  of	  platelet	  transfusions	  
o Hospital	  admissions/days	  for	  neutropenia	  with	  fever	  or	  infection	  
o Highest	  value	  for	  serum	  ALT	  and	  total	  bilirubin	  
o Grade	  3	  or	  4	  mucositis	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
26  
o Treatment	   delays	   or	   missed	   chemotherapy	   doses	   to	   allow	   for	   recovery	   from	  
toxicity	  
o Dose	  modifications	  for	  toxicity	  
o Deaths	  attributed	  to	  chemotherapy	  toxicity	  
• After	  the	  first	  12	  patients	  have	  completed	  therapy,	  the	  incidence	  and	  severity	  of	  the	  
events	   listed	   above	   as	   well	   as	   the	   nephrotoxicity	   and	   ototoxicity	   endpoints	   will	   be	  
assessed	  by	  Cycle	  according	  to	  HDMTX	  infusion	  duration	  (4	  h	  vs.	  12	  h)	  and	  according	  to	  
whether	   or	   not	   IV	   pantoprazole	   was	   administered	   with	   cisplatin	   to	   ensure	   that	   the	  
experimental	  dosing	  methods	  are	  not	  associated	  with	  excessive	  toxicity.	  
• After	   the	   first	   12	   patients	   have	   undergone	   resection	   of	   their	   primary	   tumor,	   the	  
radiographic	  response	  (log	  ratio)	  and	  histologic	  response	  (%	  necrosis)	  will	  be	  assessed	  
according	  to	  HDMTX	  infusion	  duration	  (4	  h	  vs.	  12	  h)	  during	  Cycles	  1	  &	  2	  and	  according	  to	  
whether	  or	  not	  IV	  pantoprazole	  was	  administered	  with	  cisplatin	  during	  Cycles	  1	  &	  2	  to	  
ensure	   that	   the	   anti-­‐tumor	   effect	   of	   the	   MAP	   chemotherapy	   regimen	   is	   not	  
compromised	  by	  the	  experimental	  dosing	  methods.	  	  
6.	   STATISTICAL	  CONSIDERATIONS	  
This	   is	   a	   pilot	   study	   to	   evaluate	   pharmacologically-­‐based	   dosing	   approaches	   to	   prevent	   the	  
nephrotoxic	   effects	   of	   HDMTX	   and	   cisplatin	   and	   ototoxic	   effect	   of	   cisplatin	   in	   children,	  
adolescents	   and	   young	   adults	   with	   osteosarcoma	   treated	   with	   the	   standard	   MAP	  
chemotherapy	  regimen.	  Acute	  biomarkers	  of	  AKI	  (KIM-­‐1,	  NAG,	  NGAL)	  and	  renal	  function	  tests	  
(creatinine,	  cystatin	  C,	  FEMg)	  will	  be	  used	  to	  assess	  the	  incidence	  and	  severity	  of	  nephrotoxicity	  
after	  each	  course	  of	  cisplatin	  (with	  and	  without	  pantoprazole)	  and	  HDMTX	  (infused	  over	  4	  and	  
12	  h),	  and	  audiograms	  will	  be	  used	  to	  measure	  hearing	  loss	  prior	  to	  cycles	  1-­‐4	  and	  at	  the	  end	  of	  
therapy.	  
A	  2	  x	  2	  factorial,	  randomized	  crossover	  design	  will	  allow	  patients	  to	  serve	  as	  their	  own	  control.	  
Nephrotoxicity	   data	   will	   be	   analyzed	   by	   course	   for	   cisplatin	   and	   by	   dose	   for	   HDMTX,	   and	  
ototoxicity	   data	   will	   be	   analyzed	   by	   treatment	   cycle.	   All	   endpoints	   will	   be	   analyzed	   as	  
continuous	  variables.	  
6.1	   Statistical	  Analysis	  Plan	  
6.1.1	   Primary	  Endpoints	  
• Urinary	   KIM-­‐1,	   NAG	   and	   NGAL	   will	   be	   normalized	   to	   the	   urinary	   creatinine	  
concentration	  measured	  in	  the	  same	  urine	  specimen	  and	  expressed	  as	  ng/mg	  Cr.	  The	  
end	  of	  infusion	  and	  7-­‐day	  post	  values	  for	  each	  biomarker	  will	  be	  analyzed	  by	  course	  for	  
cisplatin	  with	  vs.	  without	  pantoprazole	  and	  by	  dose	  for	  HDMTX	  administered	  as	  a	  4	  h	  vs.	  
12	  h	  infusion.	  
• GFR	  will	  be	  estimated	  from	  serum	  creatinine	  and	  from	  the	  cystatin	  C	  (see	  Section	  1.3.3)	  
prior	  to	  each	  course	  of	  cisplatin	  and	  each	  dose	  of	  HDMTX	  and	  7-­‐days	  post	  treatment.	  A	  
decrease	   in	   the	   GFR	   will	   reflect	   toxicity	   from	   the	   immediately	   preceding	   course	   of	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
27  
cisplatin	  or	  dose	  of	  HDMTX.	  Change	  in	  GFR	  will	  be	  analyzed	  by	  course	  for	  cisplatin	  with	  
vs.	  without	  pantoprazole	  and	  by	  dose	  for	  HDMTX	  administered	  as	  a	  4	  h	  vs.	  12	  h	  infusion.	  
• Change	  in	  FEMg	  from	  pre-­‐dose	  1	  (day	  1	  of	  cycles	  1-­‐4)	  of	  cisplatin	  to	  pre-­‐dose	  2	  (day	  2	  of	  
cycles	  1-­‐4)	  of	  cisplatin	  and	  day	  7	  will	  be	  measured	  for	  each	  cycle	  containing	  cisplatin	  
(n=4).	   Change	   in	   FEMg	   will	   be	   analyzed	   by	   course	   for	   cisplatin	   with	   vs.	   without	  
pantoprazole	  to	  assess	  renal	  tubular	  toxicity.	  
• The	  urinary	  MTX	  concentration	  at	  the	  end	  of	  each	  HDMTX	  infusion	  on	  cycles	  1-­‐4	  will	  be	  
tabulated	  for	  the	  4	  and	  12	  h	  infusion	  durations	  and	  compared	  to	  the	  MTX	  solubility	  limit	  
for	  the	  urine	  pH	  to	  determine	  whether	  the	  solubility	  limit	  was	  exceeded.	  End	  infusion	  
urine	  MTX	  concentration	  will	  also	  be	  correlated	  with	  simultaneous	  serum	  concentration	  
and	   with	   the	   severity	   of	   renal	   toxicity	   as	   measured	   by	   AKI	   biomarkers	   and	   serum	  
creatinine	  and	  serum	  cystatin	  C.	  
• An	  audiogram	  will	  be	  performed	  prior	  to	  Cycles	  1,	  2,	  3	  and	  4	  and	  at	  the	  end	  of	  therapy	  
(after	  cycle	  6).	  The	  average	  hearing	  level	  (HL)	  threshold	  in	  dB	  over	  the	  frequency	  range	  
of	   4,000-­‐8,000	   Hz	   will	   be	   derived	   separately	   for	   each	   ear	   from	   each	   audiogram.	   The	  
increase	  in	  the	  average	  HL	  threshold	  in	  dB	  from	  the	  pre-­‐cycle	  1	  to	  pre-­‐cycle	  2,	  from	  pre-­‐
cycle	  2	  to	  pre-­‐cycle	  3,	  from	  pre-­‐cycle	  3	  to	  pre-­‐cycle	  4,	  and	  from	  pre-­‐cycle	  4	  to	  end	  of	  
therapy	  audiograms	  will	  be	  derived	  and	  this	  increase	  in	  HL	  threshold	  will	  be	  compared	  
for	  cisplatin	  with	  vs.	  without	  pantoprazole.	  
For	  each	  primary	  endpoint,	  we	  will	  construct	  separate	  models	  for	  comparison	  of	  cisplatin	  
with	  vs.	  without	  pantoprazole	  and	  for	  comparison	  of	  the	  4	  vs.	  12	  h	  infusion	  durations	  of	  
HDMTX.	   Linear	   mixed	   effects	   model	   will	   be	   used	   for	   this	   crossover	   design,	   including	   all	  
repeated	  measures	  as	  the	  outcome,	  order	  type	  and	  period	  number	  as	  the	  fixed	  effects,	  and	  
a	  subject	  level	  random	  intercept	  to	  account	  for	  within-­‐subject	  correlation	  (or	  unstructured	  
correlation	   matrix	   for	   the	   repeated	   measures	   if	   needed).	   Appropriate	   contrasts	   will	   be	  
constructed	  to	  test	  for	  treatment	  effect.	  For	  nephrotoxicity	  outcomes	  using	  AKI	  biomarkers,	  
little	  carryover	  effect	  is	  expected.	  For	  ototoxicity	  outcomes,	  carryover	  effect	  is	  possible,	  but	  
unlikely	  to	  be	  differential	  between	  comparison	  arms	  because	  we	  randomized	  the	  order	  of	  
treatment.	  Nevertheless,	  we	  will	  conduct	  a	  statistical	  test	  for	  differential	  carryover	  effect,	  
acknowledging	   limited	   power	   with	   this	   test.	   If	   differential	   carryover	   is	   present	   in	   the	  
analysis	  we	  will	  only	  use	  the	  data	  from	  cycle	  1.	  
6.1.2	   Secondary	  Endpoints	  
• Common	   grade	   ≥2	   toxicities	   from	   cisplatin	   +	   doxorubicin	   (other	   than	   cisplatin	  
nephrotoxicity	   and	   ototoxicity),	   including	   myelosuppression,	   mucositis,	   nausea	   and	  
vomiting	   leading	   to	   dehydration	   requiring	   IV	   fluid	   hydration,	   peripheral	   neuropathy,	  
cardiac	  dysfunction,	  and	  treatment	  delays	  due	  to	  toxicity	  will	  be	  collected	  and	  graded	  
according	   to	   CTCAE	   v.4.	   Similarly,	   HDMTX-­‐related	   grade	   ≥2	   toxicities	   (other	   than	  
nephrotoxicity),	   including	   myelosuppression,	   mucositis,	   dermatitis,	   hepatotoxicity	   and	  
neurotoxicity	   will	   be	   collected	   and	   graded.	   The	   incidence	   and	   severity	   (grade)	   of	  
toxicities	   will	   be	   tabulated	   and	   compared	   by	   treatment	   arm	   (with	   vs.	   without	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
28  
pantoprazole	  for	  cisplatin	  +	  doxorubicin	  and	  by	  infusion	  duration	  for	  HDMTX).	  
• Response	   to	   2	   cycles	   of	   neoadjuvant	   chemotherapy	   will	   be	   assessed	   radiographically	  
and	  histologically.	  
o The	   volume	   of	   the	   tumor	   on	   MRI	   will	   be	   measured	   pretreatment	   and	   pre-­‐
operatively	  (post-­‐cycle	  2).	  The	  log	  ratio	  (Section	  5.2)	  of	  the	  two	  measurements	  
will	  be	  derived	  and	  compared	  using	  a	  non-­‐parametric	  analysis	  for	  cisplatin	  with	  
(patients	  on	  treatment	  arms	  1	  and	  3)	  vs.	  without	  (patients	  on	  treatment	  arms	  2	  
and	  4)	  pantoprazole	  and	  for	  HDMTX	  administered	  as	  a	  4	  h	  infusion	  (treatment	  
arms	  1	  and	  2)	  vs.	  a	  12	  h	  infusion	  (treatment	  arms	  3	  and	  4).	  
o The	   tumor	   specimen	   that	   is	   removed	   after	   2	   cycles	   of	   neoadjuvant	  
chemotherapy	  will	  be	  evaluated	  histologically	  and	  the	  percent	  of	  the	  tumor	  that	  
is	   necrotic	   will	   be	   estimated	   from	   review	   of	   multiple	   sections.	   The	   percent	  
necrosis	   will	   be	   compared	   using	   a	   non-­‐parametric	   analysis	   for	   cisplatin	   with	  
(patients	  on	  treatment	  arms	  1	  and	  3)	  vs.	  without	  (patients	  on	  treatment	  arms	  2	  
and	  4)	  pantoprazole	  and	  for	  HDMTX	  administered	  as	  a	  4	  h	  infusion	  (treatment	  
arms	  1	  and	  2)	  vs.	  a	  12	  h	  infusion	  (treatment	  arms	  3	  and	  4.).	  
• GFR	   estimated	   from	   serum	   creatinine	   using	   the	   modified	   Schwartz	   formula	   will	   be	  
correlated	  with	  GFR	  estimated	  from	  serum	  cystatin	  C	  (Section	  1.3.3,	  the	  equation	  also	  
includes	   serum	   creatinine	   and	   BUN).	   The	   presence	   and	   amount	   (normalized	   to	  
creatinine	   concentration)	   of	   biomarkers	   of	   AKI	   in	   urine	   will	   be	   correlated	   with	  
proteinuria,	  serum	  creatinine,	  and	  change	  in	  FEMg.	  
• BSAP	   levels	   will	   be	   correlated	   with	   tumor	   volume	   and	   presence	   or	   absence	   of	  
metastatic	  disease	  at	  diagnosis.	  BSAP	  after	  2	  cycles	  of	  neoadjuvant	  chemotherapy	  will	  
be	  correlated	  with	  tumor	  volume	  and	  percent	  tumor	  necrosis	  post-­‐cycle	  2.	  BSAP	  drawn	  
post-­‐operatively,	  at	  the	  end	  of	  therapy	  and	  at	  the	  time	  of	  relapse	  will	  be	  correlated	  with	  
disease	  status.	  
• An	   osteosarcoma-­‐specific	   Patient	   Reported	   Outcome	   survey	   will	   be	   used	   to	   assess	  
disease-­‐related	  and	  treatment-­‐related	  symptoms	  and	  tested	  in	  the	  patients	  enrolled	  on	  
this	   trial.	   The	   patient	   reported	   outcomes	   will	   be	   compared	   to	   toxicity	   data	   collected	  
from	  the	  medical	  record	  and	  to	  physician	  assessments	  of	  patient	  status	  (performance	  
score).	  
6.2	   Sample	  Size	  and	  Power	  
The	   primary	   endpoints	   are	   sensitive	   urinary	   biomarkers	   of	   AKI	   and	   audiograms,	   and	   all	  
endpoints	  are	  continuous	  variables.	  The	  study	  is	  designed	  to	  detect	  a	  signal	  sufficient	  to	  justify	  
a	  randomized	  trial	  that	  would	  be	  conducted	  in	  a	  cooperative	  group	  setting.	  
Twenty-­‐four	  patients	  will	  be	  enrolled	  and	  data	  will	  be	  analyzed	  by	  dose	  or	  treatment	  cycle	  with	  
patients	  serving	  as	  their	  own	  control.	  Over	  the	  past	  5	  years	  57	  newly	  diagnosed	  patients	  with	  
osteosarcoma	  were	  seen	  and	  treated	  at	  CHOP.	  This	  is	  an	  average	  of	  11.5	  patients	  per	  year,	  
indicating	  that	  we	  can	  complete	  accrual	  to	  this	  study	  in	  about	  two	  years.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
29  
• For	   the	   analyses	   of	   nephrotoxicity	   using	   biomarker	   endpoints	   of	   AKI	   (Urinary	   KIM-­‐1,	  
NAG	  and	  NGAL),	  data	  will	  be	  the	  analyzed	  by	  course	  or	  by	  dose.	  Each	  patient	  will	  have	  2	  
courses	   of	   cisplatin	   with	   pantoprazole	   and	   2	   courses	   without	   pantoprazole.	   A	   power	  
calculation	   was	   conducted	   under	   the	   framework	   of	   a	   multivariate	   general	   linear	  
hypothesis	  for	  general	  linear	  models,	  using	  the	  Wilks	  Lambda	  test	  with	  a	  significance	  
level	  of	  0.05.110,111	  Because	  the	  comparison	  will	  be	  within	  subject,	  a	  larger	  within-­‐subject	  
correlation	   will	   provide	   greater	   power	   for	   the	   test.	   Based	   on	   the	   most	   conservative	  
assumption	  that	  all	  repeated	  measures	  are	  independent,	  we	  will	  have	  80%	  power	  to	  
detect	   a	   0.60SD	   difference	   for	   with	   vs.	   without	   pantoprazole.	   Assuming	   a	   moderate	  
correlation	  of	  0.5,	  we	  will	  have	  80%	  power	  to	  detect	  a	  smaller	  difference	  of	  0.42SD.	  
Variability	   (SD)	   for	   these	   biomarkers	   in	   children	   with	   AKI	   are	   not	   available,	   but	   in	   a	  
healthy	  adult	  population,112	  the	  mean	  value	  of	  KIM-­‐1	  was	  0.228	  with	  a	  SD	  of	  0.094,	  so	  
assuming	   a	   similar	   mean	   and	   SD	   for	   pediatric	   patients,	   we	   will	   have	   80%	   power	   to	  
detect	   a	   0.056	   difference	   (about	   25%	   decrease)	   in	   the	   mean	   KIM-­‐1	   value	   with	  
pantoprazole,	   assuming	   independence	   among	   repeated	   measures.	   We	   will	   have	   80%	  
power	   to	   detect	   a	   difference	   of	   0.039	   (about	   17%	   decrease)	   if	   the	   correlation	   is	  
moderate	  (e.g.	  0.5).	  	  
• For	  comparison	  of	  HDMTX	  infusion	  durations,	  each	  patient	  will	  have	  4	  doses	  with	  4	  h	  
infusion	  duration	  and	  4	  doses	  with	  12	  h	  infusion	  duration.	  Using	  the	  Wilks	  Lambda	  test	  
with	  a	  significance	  level	  of	  0.05	  under	  the	  framework	  of	  general	  linear	  models,	  with	  24	  
subjects	  of	  8	  repeated	  measures	  (4	  measures	  with	  4	  h	  duration	  and	  4	  measures	  with	  12	  
h	  duration),	  we	  will	  have	  80%	  power	  to	  detect	  a	  0.42SD	  difference	  assuming	  a	  within-­‐
subject	  correlation	  of	  0,	  and	  will	  detect	  a	  0.30SD	  difference	  assuming	  a	  correlation	  of	  
0.5.	  
• For	  the	  analyses	  of	  ototoxicity	  data	  (hearing	  level	  thresholds	  from	  4	  to	  8	  kHz	  in	  dB),	  
treatment	   cycle	   will	   be	   the	   analysis	   unit.	   Each	   patient	   will	   have	   2	   cycles	   with	  
pantoprazole	  and	  2	  cycles	  without	  pantoprazole.	  Using	  the	  Wilks	  Lambda	  test	  with	  a	  
significance	  level	  of	  0.05	  under	  the	  framework	  of	  general	  linear	  models,	  with	  24	  subjects	  
of	  4	  repeated	  measures	  (2	  measures	  with	  and	  2	  measures	  without	  pantoprazole),	  we	  
will	  have	  80%	  power	  to	  detect	  a	  0.60SD	  difference	  assuming	  a	  within-­‐subject	  correlation	  
of	  0,	  and	  will	  detect	  a	  0.42SD	  difference	  assuming	  a	  correlation	  of	  0.5.	  	  Moreover,	  if	  
differential	  carryover	  is	  present,	  we	  will	  only	  use	  the	  data	  from	  the	  first	  two	  cycles,	  so	  
that	   the	   comparison	   will	   be	   between	   subjects,	   with	   12	   subjects	   in	   each	   comparison	  
group.	  We	  will	  then	  have	  80%	  power	  to	  detect	  a	  1.2SD	  difference,	  based	  on	  a	  two-­‐sided,	  
two-­‐sample	  t	  test	  with	  a	  significance	  level	  of	  0.05.	  
6.3	   Interim	  Analysis	  
The	  sample	  size	  in	  this	  pilot	  study	  is	  too	  small	  to	  derive	  a	  statistically	  valid	  stopping	  rule	  with	  
sufficient	   sensitivity.	   However,	   after	   the	   first	   12	   patients	   have	   completed	   treatment,	   the	  
investigators	   will	   evaluate	   the	   following	   parameters	   to	   determine	   whether	   pantoprazole	   or	  
prolonging	  the	  HDMTX	  infusion	  affect	  toxicity	  or	  anti-­‐tumor	  response	  to	  MAP:	  
• The	   incidence	   and	   severity	   (CTCAE	   grade)	   of	   toxicities	   will	   be	   compared	   for	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
30  
cisplatin/doxorubicin	  with	  vs.	  without	  pantoprazole	  and	  for	  HDMTX	  infused	  over	  4	  h	  vs.	  
12	  h	  within	  and	  across	  patients.	  
• Radiographic	  response	  (log	  ratio)	  and	  histological	  response	  (%	  tumor	  necrosis)	  following	  
2	   cycles	   of	   neoadjuvant	   chemotherapy	   will	   be	   compared	   in	   patients	   who	   received	  
pantoprazole	   vs.	   patients	   who	   did	   not	   receive	   pantoprazole	   and	   in	   patients	   who	  
received	  HDMTX	  over	  4	  h	  vs.	  12	  h.	  
If	   the	   toxicity	   from	   MAP	   is	   clinically	   significantly	   worse	   when	   cisplatin	   is	   administered	   with	  
pantoprazole	  or	  when	  HDMTX	  is	  infused	  over	  12	  h	  OR	  if	  radiographic	  or	  histological	  response	  to	  
neoadjuvant	  chemotherapy	  is	  clinically	  significantly	  less	  in	  patients	  receiving	  pantoprazole	  or	  a	  
prolonged	  HDMTX	  infusion,	  then	  the	  intervention	  found	  to	  have	  the	  worse	  outcome	  will	  be	  
discontinued.	  
7.	   STUDY	  MEDICATIONS	  
All	  medications	  prescribed	  in	  this	  protocol	  are	  commercially	  available.	  The	  MAP	  chemotherapy	  
regimen,	  which	  includes	  cisplatin,	  doxorubicin	  and	  methotrexate,	  is	  the	  standard	  of	  care	  for	  
osteosarcoma,	  and	  the	  doses,	  routes	  of	  administration	  and	  schedules	  are	  also	  standard,	  except	  
for	  the	  alternative	  infusion	  duration	  for	  HDMTX.	  Intravenous	  pantoprazole	  is	  being	  studied	  to	  
determine	   whether	   co-­‐administration	   with	   cisplatin	   can	   selectively	   prevent	   cisplatin	  
nephrotoxicity	  and	  ototoxicity.	  
7.1	   Intravenous	  Pantoprazole	  (PROTONIX	  I.V.)	  
• IUPAC	   Name:	   (RS)-­‐6-­‐(Difluoromethoxy)-­‐2-­‐[(3,4-­‐dimethoxypyridin-­‐2-­‐yl)methylsulfinyl]-­‐
1H-­‐benzo[d]imidazole	  
• Molecular	  formula	  &	  weight:	  C16H15F2N3O4S	  -­‐	  383.4	  g/mol	  (Fig	  5,	  chemical	  structure)	  
• Manufacturer:	  Pfizer	  Injectables	  (Distributed	  by	  Wyeth	  Pharmaceuticals	  Inc.)	  
• Drug	  supply:	  purchased	  from	  commercial	  sources	  
• Indications:	  gastroesophageal	  reflux	  associated	  with	  a	  history	  of	  erosive	  esophagitis	  and	  
pathological	  hypersecretory	  conditions	  (e.g.,	  Zollinger-­‐Ellison	  syndrome)	  
• Dosage	   forms	   and	   strength:	  supplied	  in	  a	  vial	  containing	  40	  mg	  of	  pantoprazole	  as	  a	  
freeze	  dried	  powder	  
• Storage	   and	   Handling:	   store	   vials	   at	   20-­‐25°C	   (USP	   controlled	   room	   temperature);	  
excursions	  permitted	  to	  15-­‐30°C.	  
• Dosing	   (Section	   3.1.3):	   0.3	   mg/kg	   IV	   over	   15	   min	   immediately	   prior	   to	   each	   dose	   of	  
cisplatin	  as	  a	  loading	  dose	  followed	  by	  1.3	  mg/kg	  IV	  infused	  over	  4	  h	  concurrent	  with	  
each	  4	  h	  cisplatin	  infusion	  on	  either	  Cycles	  1	  &	  2	  OR	  Cycles	  3	  &	  4	  depending	  on	  the	  
treatment	   arm.	   See	   Section	   1.3.8	   for	   the	   rationale	   for	   pantoprazole	   administration	  
schedule.	  
• Compatibility:	  the	  compatibility	  of	  the	  cisplatin	  and	  pantoprazole	  drugs	  solutions	  was	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
31  
tested	   at	   UPenn	   by	   Dr.	   Suzanne	   Wehrli,	   and	   at	   the	   concentrations	   used	   for	   drug	  
administration,	   there	   was	   no	   evidence	   of	   an	   interaction	   –	   indicating	   that	   the	  
pantoprazole	  solution	  can	  be	  infused	  into	  the	  same	  intravenous	  tubing	  through	  which	  
the	  cisplatin	  is	  being	  infused.	  
• Clinical	   pharmacology:	   see	   Section	   1.3.5	   for	   pediatric	   pharmacokinetic	   data	   and	  
tolerability	  of	  single	  doses	  in	  children	  
• Adverse	  reactions:	  reactions	  reported	  in	  clinical	  trials	  in	  adults	  (n=1473)	  with	  GERD	  at	  a	  
frequency	  >2%	  included	  headache	  (12%),	  diarrhea	  (8.8%),	  nausea	  (7%),	  abdominal	  pain	  
(6.2%),	  vomiting	  (4.3%),	  flatulence	  (3.9%),	  dizziness	  (3%),	  arthralgia	  (2.8%).	  The	  use	  of	  
PPIs	  is	  associated	  with	  a	  moderately	  increased	  risk	  of	  C.	  difficile	  colitis.113,114	  
• Drug	  accountability:	  a	  separate	  drug	  supply	  will	  be	  purchased	  for	  this	  study	  using	  the	  
research	  funds,	  and	  NCI	  DARFs	  will	  be	  used	  to	  track	  drug	  supply.	  
• IND	   status:	  this	  pilot	  study	  will	  evaluate	  pantoprazole	  for	  a	  non-­‐approved	  indication.	  
The	  use	  of	  pantoprazole	  in	  this	  study	  meets	  the	  five	  criteria	  for	  IND	  exempt	  status:	  
o The	   study	   is	   not	   intended	   to	   support	   FDA	   approval	   of	   a	   new	   indication	   or	   a	  
significant	  change	  in	  the	  product	  labeling	  
o The	  study	  is	  not	  intended	  to	  support	  a	  significant	  change	  in	  the	  advertising	  for	  
the	  product	  
o The	  investigation	  does	  not	  involve	  a	  route	  of	  administration	  or	  dosage	  level	  or	  
use	  in	  a	  patient	  population	  or	  other	  factor	  that	  significantly	  increases	  the	  risk	  (or	  
decreases	   the	   acceptability	   of	   the	   risks)	   associated	   with	   the	   use	   of	   the	   drug	  
product	  
o The	  study	  is	  conducted	  in	  compliance	  with	  institutional	  review	  board	  (IRB)	  and	  
informed	  consent	  regulations	  set	  forth	  in	  21	  CFR	  parts	  56	  and	  50	  
o The	  study	  is	  conducted	  in	  compliance	  with	  §	  312.7	  (promotion	  and	  charging	  for	  
investigational	  drugs)	  
8.	   SAFETY	  MANAGEMENT	  
Patients	   with	   osteosarcoma	   that	   will	   be	   treated	   with	   surgery	   (or	   radiation	   if	   surgery	   is	   not	  
feasible)	  and	  a	  standard	  3-­‐drug	  MAP	  chemotherapy	  will	  be	  enrolled	  on	  this	  pilot	  trial	  to	  assess	  
two	   interventions	   designed	   to	   reduce	   the	   nephrotoxicity	   and	   ototoxicity	   from	   cisplatin	   and	  
HDMTX.	  The	  primary	  endpoints	  on	  this	  trial	  will	  monitor	  the	  incidence	  and	  severity	  of	  these	  
known	  toxicities.	  Secondary	  endpoints	  include	  monitoring	  the	  overall	  toxicity	  of	  the	  regimen	  
and	  response	  to	  neoadjuvant	  chemotherapy	  to	  determine	  whether	  the	  interventions	  increase	  
the	  incidence	  or	  severity	  of	  the	  other	  expected	  toxicities	  of	  the	  regimen	  or	  alter	  the	  response	  to	  
neoadjuvant	  treatment.	  Toxicities	  of	  the	  regimen	  will	  be	  categorized	  and	  graded	  according	  to	  
the	  standard	  NCI	  CTCAE	  v.4	  toxicity	  criteria.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
32  
8.1	   Clinical	  adverse	  events	  
In	  addition	  to	  nephrotoxicity	  and	  ototoxicity,	  the	  MAP	  chemotherapy	  regimen	  is	  expected	  to	  
cause	   substantial	   toxicity	   in	   all	   patients	   undergoing	   treatment	   for	   osteosarcoma,	   including	  
myelosuppression,	  febrile	  neutropenia	  and	  infection,	  nausea	  and	  vomiting,	  mucositis,	  anorexia,	  
weight	  loss,	  fatigue,	  hepatotoxicity,	  and	  neurotoxicity.	  
Clinical	  adverse	  events	  (AEs)	  related	  to	  the	  research	  interventions,	  which	  are	  administration	  of	  
pantoprazole	  with	  cisplatin	  and	  prolonging	  the	  infusion	  duration	  of	  HDMTX,	  will	  be	  monitored	  
throughout	  this	  study.	  	  
8.2	   Adverse	  Event	  Reporting	  
SAEs	  will	  be	  promptly	  reported	  to	  the	  IRB	  in	  accordance	  with	  CHOP	  IRB	  policies.	  AEs	  that	  are	  
not	  serious	  but	  that	  might	  involve	  risks	  to	  subjects	  will	  be	  summarized	  in	  narrative	  or	  other	  
format	  and	  submitted	  to	  the	  IRB	  at	  the	  time	  of	  continuing	  review.	  
8.3	   Definition	  of	  an	  Adverse	  Event	  
An	   adverse	   event	   is	   any	   untoward	   medical	   occurrence	   in	   a	   subject	   who	   has	   received	   an	  
intervention	  (drug,	  biologic,	  or	  other	  intervention).	  The	  occurrence	  does	  not	  necessarily	  have	  to	  
have	   a	   causal	   relationship	   with	   the	   treatment.	   An	   AE	   can	   therefore	   be	   any	   unfavorable	   or	  
unintended	  sign	  (including	  an	  abnormal	  laboratory	  finding,	  for	  example),	  symptom,	  or	  disease	  
temporally	  associated	  with	  the	  use	  of	  a	  medicinal	  product,	  whether	  or	  not	  considered	  related	  
to	  the	  medicinal	  product.	  
AEs	  (including	  SAEs)	  that	  are	  grade	  ≥2	  in	  severity	  and	  related	  to	  the	  treatment	  will	  be	  collected	  
in	   an	   eCRF	   in	   OnCore	   including	   CTCAE	   category,	   grade,	   date	   of	   onset,	   duration,	   attribution	  
(causality),	  and	  outcome	  of	  the	  event	  (resolution).	  
8.4	   Definition	  of	  a	  Serious	  Adverse	  Event	  (SAE)	  
An	  SAE	  is	  any	  adverse	  drug	  experience	  occurring	  at	  any	  dose	  that	  results	  in	  any	  of	  the	  following	  
outcomes:	  	  
• death,	  
• a	  life-­‐threatening	  event	  (at	  risk	  of	  death	  at	  the	  time	  of	  the	  event),	  	  
• requires	  inpatient	  hospitalization	  or	  prolongation	  of	  existing	  hospitalization,	  
• a	  persistent	  or	  significant	  disability/incapacity,	  or	  
• a	  congenital	  anomaly/birth	  defect	  in	  the	  offspring	  of	  a	  subject.	  	  	  
Important	   medical	   events	   that	   may	   not	   result	   in	   death,	   be	   life-­‐threatening,	   or	   require	  
hospitalization	  may	  be	  considered	  a	  serious	  adverse	  drug	  event	  when,	  based	  upon	  appropriate	  
medical	   judgment,	   they	   may	   jeopardize	   the	   subject	   and	   may	   require	   medical	   or	   surgical	  
intervention	  to	  prevent	  one	  of	  the	  outcomes	  listed	  in	  this	  definition.	  
A	  distinction	  should	  be	  drawn	  between	  serious	  and	  severe	  AEs.	  A	  severe	  AE	  is	  a	  major	  event	  of	  
its	  type.	  A	  severe	  AE	  does	  not	  necessarily	  need	  to	  be	  considered	  serious.	  For	  example,	  nausea	  
that	  persists	  for	  several	  hours	  may	  be	  considered	  severe	  nausea,	  but	  would	  not	  be	  an	  SAE.	  On	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
33  
the	  other	  hand,	  a	  stroke	  that	  results	  in	  only	  a	  limited	  degree	  of	  disability	  may	  be	  considered	  a	  
mild	  stroke,	  but	  would	  be	  an	  SAE.	  	  
8.4.1	   Relationship	  of	  SAE	  to	  study	  drug	  or	  other	  intervention	  or	  procedure	  
The	  relationship	  of	  each	  SAE	  to	  the	  study	  intervention	  will	  be	  characterized	  using	  one	  of	  the	  
following	   terms	   in	   accordance	   with	   CHOP	   IRB	   Guidelines:	   definitely,	   probably,	   possibly,	  
unlikely	  or	  unrelated.	  	  
8.4.2	   IRB/IEC	  Notification	  of	  SAEs	  
The	  IRB	  will	  be	  promptly	  notified	  of	  all	  on-­‐site	  AEs	  that	  are	  (1)	  serious,	  (2)	  unexpected	  and	  
(3)	   related	   to	   the	   research	   procedures.	   Other	   unanticipated	   problems	   involving	   risk	   to	  
subjects	   or	   others	   will	   also	   be	   reported	   promptly	   using	   the	   CHOP	   Internal	   SAE	   reporting	  
form	  and	  in	  accordance	  with	  the	  following	  timeline.	  	  
Type	  of	  Internal	  Adverse	  
Event	  
Initial	  Notification	  
(Phone,	  Email,	  Fax)	  
Written	  Report	  
Internal	  (on-­‐site)	  SAEs	  	  
Death	  or	  Life	  Threatening	  	  
24	  hours	  
Within	  2	  calendar	  days	  
Internal	  (on-­‐site)	  SAEs	  	  
All	  other	  SAEs	  
7	  days	  
Within	  7	  business	  days	  
Unanticipated	  Problems	  
Related	  to	  Research	  
7	  days	  	  
Within	  7	  business	  days	  
All	  other	  AEs	  
N/A	  
Summary	   of	   AEs	   Reported	   at	  
Time	  of	  Continuing	  Review	  
8.4.3	   Follow-­‐up	  report	  
If	  an	  SAE	  has	  not	  resolved	  at	  the	  time	  of	  the	  initial	  report,	  a	  follow-­‐up	  report	  including	  all	  
relevant	   new	   or	   reassessed	   information	   (e.g.,	   concomitant	   medication,	   medical	   history)	  
should	  be	  submitted	  to	  the	  IRB.	  All	  SAE	  should	  be	  followed	  until	  either	  resolved	  or	  stable.	  	  
9.	   STUDY	  ADMINISTRATION	  
9.1	   Data	  collection	  and	  management	  
9.1.1	   Confidentiality	  
Demographic	  data	  including	  each	  patient’s	  name,	  MRN,	  DOB,	  and	  a	  unique	  patient	  study	  
number	   is	   entered	   and	   stored	   in	   Oncore	   CRM	   (Forte	   Research),	   the	   CCCR’s	   clinical	   trial	  
management	   system.	   Data	   regarding	   markers	   of	   Acute	   Kidney	   Injury	   (Cystatin-­‐C,	   KIM-­‐1,	  
NGAL,	   NAG)	   will	   be	   collected	   using	   secure	   OnCore	   electronic	   case	   report	   forms	   (eCRFs).	  
Results	   of	   standard	   renal	   function	   tests,	   audiograms,	   bone-­‐specific	   alkaline	   phosphatase	  
assays,	  nutritional	  assessments,	  and	  response	  assessments	  (radiographic	  and	  histologic)	  as	  
well	  as	  toxicity	  data	  will	  also	  be	  entered	  into	  Oncore	  eCRFs.	  Patient	  related	  outcomes	  forms	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
34  
completed	  by	  a	  parent	  will	  be	  stored	  in	  a	  locked	  file	  cabinet	  in	  the	  CTRB.	  These	  forms	  will	  
have	  patient	  identifiers	  (name,	  DOB,	  MRN).	  
9.1.2	   Security	  
OnCore	  is	  maintained	  on	  a	  Research	  IS	  server	  that	  is	  password	  protected	  and	  backed	  up	  
frequently.	  File	  cabinets	  in	  CTRB	  with	  paper	  forms	  will	  be	  kept	  locked.	  
9.1.3	   Anonymization,	  de-­‐identification,	  or	  destruction	  
The	  PHI	  in	  OnCore	  will	  be	  kept	  indefinitely.	  	  
9.2	   Confidentiality	  
All	  data	  and	  records	  generated	  during	  this	  study	  will	  be	  kept	  confidential	  in	  accordance	  with	  
CHOP	  Institutional	  policies	  and	  HIPAA	  on	  subject	  privacy	  and	  the	  Investigator	  and	  other	  site	  
personnel	  will	  not	  use	  the	  data	  and	  records	  for	  any	  purpose	  other	  than	  conducting	  the	  study.	  
Records	   with	   identifiers	   will	   be	   retained	   indefinitely	   in	   a	   secure,	   password	   protected	  
commercial	  clinical	  trial	  management	  system	  (OnCore).	  	  
Safeguards	   to	   maintain	   subject	   confidentiality	   are	   described	   under	   Data	   Collection	   and	  
Management	  (Section	  9.1).	  
9.3	   Regulatory	  and	  ethical	  considerations	  
9.3.1	   Data	  safety	  and	  monitoring	  plan	  
The	  research	  team	  led	  by	  the	  protocol	  PI	  will	  monitor	  adverse	  events	  related	  to	  the	  protocol	  
therapy	  in	  real	  time.	  The	  progress	  of	  every	  patient	  actively	  receiving	  treatment	  on	  this	  study	  
will	  be	  discussed	  weekly	  in	  the	  Solid	  Tumor	  Team	  Meeting.	  An	  interim	  analysis	  to	  assess	  the	  
safety	   of	   the	   interventions	   will	   be	   performed	   after	   the	   first	   12	   patients	   have	   completed	  
therapy.	  
9.3.2	   Risk	  assessment	  
There	  is	  risk	  associated	  with	  the	  addition	  of	  pantoprazole	  to	  cisplatin	  therapy,	  however	  PPIs	  
are	  widely	  used	  in	  pediatric	  medicine	  and	  there	  is	  substantial	  experience	  with	  the	  toxicities	  
associated	  with	  these	  agents	  (Section	  7.1,	  Adverse	  Events).	  Single	  IV	  doses	  of	  pantoprazole	  
are	  very	  well	  tolerated	  in	  children.	  An	  association	  between	  the	  use	  of	  PPIs	  and	  C.	  difficile	  
colitis	  was	  recently	  reported,	  but	  the	  use	  of	  pantoprazole	  in	  this	  study	  is	  limited	  to	  4	  doses.	  
The	   use	   of	   cisplatin	   and	   pantoprazole	   together	   has	   not	   been	   studied	   in	   children.	   As	  
described	   in	   the	   background,	   we	   do	   not	   expect	   pantoprazole	   to	   alter	   the	   excretion	   of	  
cisplatin.	  PPIs	  are	  frequently	  administered	  chronically	  to	  patients	  receiving	  chemotherapy	  
for	   symptoms	   of	   reflux.	   The	   risk	   associated	   with	   administration	   of	   pantoprazole	   with	  
cisplatin	  must	  be	  considered	  greater	  than	  minimal.	  	  
Prolonging	   the	   infusion	   duration	   may	   alter	   the	   toxicity	   profile	   of	   HDMTX	   because	   the	  
pharmacologic	   effect	   of	   MTX	   is	   determined	   by	   the	   duration	   of	   exposure	   to	   the	   drug.	  
Leucovorin	  rescue	  will	  start	  24	  h	  after	  the	  start	  of	  the	  HDMTX	  infusion	  for	  both	  infusion	  
durations.	   The	   PK	   simulations	   (Figure	   4)	   show	   that	   plasma	   concentrations	   at	   later	   time	  
points	   are	   similar	   for	   the	   two	   infusion	   durations.	   There	   is	   considerable	   experience	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
35  
demonstrating	  tolerability	  of	  HDMTX	  in	  children	  with	  leukemia	  treated	  with	  higher	  doses	  of	  
MTX	   (e.g.,	   33	   g/m2)	   administered	   as	   prolonged	   infusions	   with	   leucovorin	   rescue.	  
Administration	   of	   12	   g/m2	   HDMTX	   infused	   over	   12	   h	   instead	   of	   4	   h	   must	   be	   considered	  
greater	  than	  minimal	  risk.	  
9.3.3	   Potential	  benefits	  of	  trial	  participation	  
The	   crossover	   design	   ensures	   all	   patients	   will	   receive	   pantoprazole	   and	   the	   prolonged	  
infusions	   of	   HDMTX.	   Therefore,	   all	   patients	   have	   the	   potential	   for	   direct	   benefit	   from	  
participation	  in	  this	  study.	  	  Pantoprazole	  may	  mitigate	  the	  nephrotoxicity	  and	  hearing	  loss	  
associated	  with	  cisplatin	  exposure.	  Prolonged	  administration	  of	  methotrexate	  may	  decrease	  
the	  risk	  of	  acute	  kidney	  injury	  in	  patients	  receiving	  this	  agent.	  In	  addition	  results	  from	  this	  
study	   could	   benefit	   future	   patients	   with	   osteosarcoma	   and	   other	   cancers	   treated	   with	  
cisplatin	   and	   HDMTX	   if	   these	   more	   rational	   dosing	   methods	   and	   sensitive	   biomarkers	   of	  
toxicity	  and	  response	  prove	  to	  be	  better.	  	  
9.3.4	   Risk-­‐benefit	  assessment	  
This	  study	  is	  associated	  with	  greater	  than	  minimal	  risk,	  however	  there	  is	  the	  prospect	  of	  
direct	  benefit	  to	  the	  subjects.	  The	  results	  of	  this	  study	  may	  provide	  pilot	  data	  for	  a	  larger	  
definitive	  clinical	  trial.	  There	  is	  the	  potential	  that	  such	  a	  study	  may	  result	  in	  adoption	  of	  new	  
and	   safer	   dosing	   administration	   schedules	   and	   modifications	   of	   supportive	   care	  
recommendations	  for	  children	  receiving	  methotrexate	  and	  cisplatin	  for	  osteosarcoma	  and	  
other	   cancer	   types.	   As	   a	   result,	   this	   study	   and	   a	   potential	   successor	   study	   may	   benefit	  
children	  who	  receive	  these	  agents	  in	  the	  future.	  
9.4	   Recruitment	  strategy	  
Potential	  subjects	  are	  identified	  from	  the	  list	  of	  children	  undergoing	  evaluation	  of	  a	  suspected	  
primary	  bone	  tumor.	  Investigators	  will	  ask	  each	  potential	  subject’s	  primary	  physician	  at	  CHOP	  
whether	  the	  subject	  is	  appropriate	  for	  this	  study,	  and,	  if	  so,	  the	  PI	  or	  a	  Co-­‐I	  will	  meet	  with	  the	  
family	  after	  the	  diagnosis	  of	  osteosarcoma	  is	  confirmed	  but	  prior	  to	  the	  initiation	  of	  therapy.	  
The	   PI	   or	   a	   Co-­‐I	   will	   discuss	   the	   study	   and	   obtain	   informed	   consent	   if	   a	   potential	   subject	   is	  
willing	  to	  participate.	  	  
9.5	   Informed	  consent/assent	  
The	  protocol	  PI	  or	  a	  co-­‐investigator	  will	  discuss	  participation	  in	  the	  study	  with	  each	  potential	  
subject	  or	  the	  parents	  or	  legal	  guardian	  of	  each	  potential	  subject.	  Written	  informed	  consent	  will	  
be	  obtained.	  The	  discussion	  will	  take	  place	  in	  a	  private	  room	  in	  the	  Oncology	  clinic	  or	  in	  the	  
patient’s	  room	  on	  the	  inpatient	  unit.	  	  The	  family	  (and	  patient)	  are	  allowed	  time	  to	  ask	  questions	  
and	  to	  read	  the	  informed	  consent	  form	  before	  signing.	  Participation	  in	  this	  study	  is	  entirely	  
voluntary	  and	  has	  no	  influence	  on	  whether	  the	  potential	  subject	  will	  receive	  clinical	  treatment	  
for	   osteosarcoma	   at	   CHOP.	   The	   consent	   process	   will	   be	   documented	   in	   a	   brief	   note	   in	   the	  
subject’s	   medical	   record	   and	   by	   the	   signed	   informed	   consent	   document.	   Assent	   will	   be	  
obtained	  in	  the	  presence	  of	  the	  potential	  minor	  subject’s	  parents/guardian	  when	  appropriate	  
based	   on	   the	   age	   and	   level	   of	   maturity	   and	   understanding	   of	   the	   subject.	   Assent	   will	   be	  
documented	  on	  the	  consent	  form.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
36  
9.6	   Payments	  to	  subjects/families	  
Subjects	  and	  families	  will	  not	  receive	  payments	  for	  participation	  in	  this	  trial.	  
10.	  	  PUBLICATION	  
This	   is	   a	   single	   Institution	   pilot	   study	   and	   measurement	   of	   the	   endpoints	   for	   the	   primary	  
objectives	  will	  be	  completed	  when	  the	  treatment	  is	  completed.	  The	  team	  of	  CHOP	  Investigators	  
plan	  to	  publish	  the	  results	  separately	  for	  the	  effect	  of	  prolonging	  the	  HDMTX	  infusion	  duration	  
on	   MTX	   nephrotoxicity	   and	   the	   effect	   of	   IV	   pantoprazole	   on	   cisplatin	   nephrotoxicity	   and	  
ototoxicity.	  
11.	  REFERENCES	  
1.	  
Nies	  AT,	  Hofmann	  U,	  Resch	  C,	  Schaeffeler	  E,	  Rius	  M,	  Schwab	  M.	  Proton	  pump	  inhibitors	  inhibit	  metformin	  
uptake	  by	  organic	  cation	  transporters	  (OCTs).	  PloS	  one	  2011;6:e22163.	  
2.	  
Anninga	  JK,	  Gelderblom	  H,	  Fiocco	  M,	  et	  al.	  Chemotherapeutic	  adjuvant	  treatment	  for	  osteosarcoma:	  where	  
do	  we	  stand?	  European	  journal	  of	  cancer	  2011;47:2431-­‐45.	  
3.	  
Kearns	  GL,	  Blumer	  J,	  Schexnayder	  S,	  et	  al.	  Single-­‐dose	  pharmacokinetics	  of	  oral	  and	  intravenous	  
pantoprazole	  in	  children	  and	  adolescents.	  Journal	  of	  clinical	  pharmacology	  2008;48:1356-­‐65.	  
4.	  
Sleijfer	  DT,	  Offerman	  JJ,	  Mulder	  NH,	  et	  al.	  The	  protective	  potential	  of	  the	  combination	  of	  verapamil	  and	  
cimetidine	  on	  cisplatin-­‐induced	  nephrotoxicity	  in	  man.	  Cancer	  1987;60:2823-­‐8.	  
5.	  
Graf	  N,	  Winkler	  K,	  Betlemovic	  M,	  Fuchs	  N,	  Bode	  U.	  Methotrexate	  pharmacokinetics	  and	  prognosis	  in	  
osteosarcoma.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  
1994;12:1443-­‐51.	  
6.	  
Jacobs	  SA,	  Stoller	  RG,	  Chabner	  BA,	  Johns	  DG.	  7-­‐Hydroxymethotrexate	  as	  a	  urinary	  metabolite	  in	  human	  
subjects	  and	  rhesus	  monkeys	  receiving	  high	  dose	  methotrexate.	  The	  Journal	  of	  clinical	  investigation	  
1976;57:534-­‐8.	  
7.	  
Link	  MP,	  Goorin	  AM,	  Miser	  AW,	  et	  al.	  The	  effect	  of	  adjuvant	  chemotherapy	  on	  relapse-­‐free	  survival	  in	  
patients	  with	  osteosarcoma	  of	  the	  extremity.	  The	  New	  England	  journal	  of	  medicine	  1986;314:1600-­‐6.	  
8.	  
Magnani	  C,	  Pastore	  G,	  Coebergh	  JW,	  Viscomi	  S,	  Spix	  C,	  Steliarova-­‐Foucher	  E.	  Trends	  in	  survival	  after	  
childhood	  cancer	  in	  Europe,	  1978-­‐1997:	  report	  from	  the	  Automated	  Childhood	  Cancer	  Information	  System	  
project	  (ACCIS).	  European	  journal	  of	  cancer	  2006;42:1981-­‐2005.	  
9.	  
Stiller	  CA,	  Passmore	  SJ,	  Kroll	  ME,	  Brownbill	  PA,	  Wallis	  JC,	  Craft	  AW.	  Patterns	  of	  care	  and	  survival	  for	  patients	  
aged	  under	  40	  years	  with	  bone	  sarcoma	  in	  Britain,	  1980-­‐1994.	  British	  journal	  of	  cancer	  2006;94:22-­‐9.	  
10.	  
Smith	  MA,	  Seibel	  NL,	  Altekruse	  SF,	  et	  al.	  Outcomes	  for	  children	  and	  adolescents	  with	  cancer:	  challenges	  for	  
the	  twenty-­‐first	  century.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  
Oncology	  2010;28:2625-­‐34.	  
11.	  
Kager	  L,	  Zoubek	  A,	  Potschger	  U,	  et	  al.	  Primary	  metastatic	  osteosarcoma:	  presentation	  and	  outcome	  of	  
patients	  treated	  on	  neoadjuvant	  Cooperative	  Osteosarcoma	  Study	  Group	  protocols.	  Journal	  of	  clinical	  
oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  2003;21:2011-­‐8.	  
12.	  
Chou	  AJ,	  Kleinerman	  ES,	  Krailo	  MD,	  et	  al.	  Addition	  of	  muramyl	  tripeptide	  to	  chemotherapy	  for	  patients	  with	  
newly	  diagnosed	  metastatic	  osteosarcoma:	  a	  report	  from	  the	  Children's	  Oncology	  Group.	  Cancer	  
2009;115:5339-­‐48.	  


	  
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	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
37  
13.	  
Meyers	  PA,	  Heller	  G,	  Healey	  JH,	  et	  al.	  Osteogenic	  sarcoma	  with	  clinically	  detectable	  metastasis	  at	  initial	  
presentation.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  
1993;11:449-­‐53.	  
14.	  
Ferrari	  S,	  Smeland	  S,	  Mercuri	  M,	  et	  al.	  Neoadjuvant	  chemotherapy	  with	  high-­‐dose	  Ifosfamide,	  high-­‐dose	  
methotrexate,	  cisplatin,	  and	  doxorubicin	  for	  patients	  with	  localized	  osteosarcoma	  of	  the	  extremity:	  a	  joint	  
study	  by	  the	  Italian	  and	  Scandinavian	  Sarcoma	  Groups.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  
American	  Society	  of	  Clinical	  Oncology	  2005;23:8845-­‐52.	  
15.	  
Goorin	  AM,	  Schwartzentruber	  DJ,	  Devidas	  M,	  et	  al.	  Presurgical	  chemotherapy	  compared	  with	  immediate	  
surgery	  and	  adjuvant	  chemotherapy	  for	  nonmetastatic	  osteosarcoma:	  Pediatric	  Oncology	  Group	  Study	  POG-­‐
8651.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  
2003;21:1574-­‐80.	  
16.	  
Lewis	  IJ,	  Nooij	  MA,	  Whelan	  J,	  et	  al.	  Improvement	  in	  histologic	  response	  but	  not	  survival	  in	  osteosarcoma	  
patients	  treated	  with	  intensified	  chemotherapy:	  a	  randomized	  phase	  III	  trial	  of	  the	  European	  Osteosarcoma	  
Intergroup.	  J	  Natl	  Cancer	  Inst	  2007;99:112-­‐28.	  
17.	  
Souhami	  RL,	  Craft	  AW,	  Van	  der	  Eijken	  JW,	  et	  al.	  Randomised	  trial	  of	  two	  regimens	  of	  chemotherapy	  in	  
operable	  osteosarcoma:	  a	  study	  of	  the	  European	  Osteosarcoma	  Intergroup.	  Lancet	  1997;350:911-­‐7.	  
18.	  
Gurney	  JG,	  Swenson	  AR,	  Bulterys	  M.	  Malignant	  Bone	  Tumors.	  In:	  Ries	  LAG,	  Smith	  MA,	  Gurney	  JG,	  et	  al.,	  eds.	  
Cancer	  Incidence	  and	  Survival:	  United	  States	  SEER	  Program	  1975-­‐1995,	  National	  Cancer	  Institute,	  SEER	  
Program.	  Bethesda,	  MD:	  NIH	  Pub.	  No.	  99-­‐4649;	  1999:99-­‐110.	  
19.	  
Bielack	  SS,	  Kempf-­‐Bielack	  B,	  Delling	  G,	  et	  al.	  Prognostic	  factors	  in	  high-­‐grade	  osteosarcoma	  of	  the	  
extremities	  or	  trunk:	  an	  analysis	  of	  1,702	  patients	  treated	  on	  neoadjuvant	  cooperative	  osteosarcoma	  study	  
group	  protocols.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  
2002;20:776-­‐90.	  
20.	  
Ritter	  J,	  Bielack	  SS.	  Osteosarcoma.	  Annals	  of	  oncology	  :	  official	  journal	  of	  the	  European	  Society	  for	  Medical	  
Oncology	  /	  ESMO	  2010;21	  Suppl	  7:vii320-­‐5.	  
21.	  
Davis	  AM,	  Bell	  RS,	  Goodwin	  PJ.	  Prognostic	  factors	  in	  osteosarcoma:	  a	  critical	  review.	  Journal	  of	  clinical	  
oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  1994;12:423-­‐31.	  
22.	  
Eilber	  F,	  Giuliano	  A,	  Eckardt	  J,	  Patterson	  K,	  Moseley	  S,	  Goodnight	  J.	  Adjuvant	  chemotherapy	  for	  
osteosarcoma:	  a	  randomized	  prospective	  trial.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  
Society	  of	  Clinical	  Oncology	  1987;5:21-­‐6.	  
23.	  
Meyers	  PA,	  Schwartz	  CL,	  Krailo	  M,	  et	  al.	  Osteosarcoma:	  a	  randomized,	  prospective	  trial	  of	  the	  addition	  of	  
ifosfamide	  and/or	  muramyl	  tripeptide	  to	  cisplatin,	  doxorubicin,	  and	  high-­‐dose	  methotrexate.	  Journal	  of	  
clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  2005;23:2004-­‐11.	  
24.	  
Meyers	  PA,	  Schwartz	  CL,	  Krailo	  MD,	  et	  al.	  Osteosarcoma:	  the	  addition	  of	  muramyl	  tripeptide	  to	  
chemotherapy	  improves	  overall	  survival-­‐-­‐a	  report	  from	  the	  Children's	  Oncology	  Group.	  Journal	  of	  clinical	  
oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  Oncology	  2008;26:633-­‐8.	  
25.	  
Ferrari	  S,	  Ruggieri	  P,	  Cefalo	  G,	  et	  al.	  Neoadjuvant	  Chemotherapy	  With	  Methotrexate,	  Cisplatin,	  and	  
Doxorubicin	  With	  or	  Without	  Ifosfamide	  in	  Nonmetastatic	  Osteosarcoma	  of	  the	  Extremity:	  An	  Italian	  
Sarcoma	  Group	  Trial	  ISG/OS-­‐1.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  
Clinical	  Oncology	  2012.	  
26.	  
Janeway	  KA,	  Grier	  HE.	  Sequelae	  of	  osteosarcoma	  medical	  therapy:	  a	  review	  of	  rare	  acute	  toxicities	  and	  late	  
effects.	  The	  lancet	  oncology	  2010;11:670-­‐8.	  
27.	  
Ferrari	  S,	  Pieretti	  F,	  Verri	  E,	  et	  al.	  Prospective	  evaluation	  of	  renal	  function	  in	  pediatric	  and	  adult	  patients	  
treated	  with	  high-­‐dose	  ifosfamide,	  cisplatin	  and	  high-­‐dose	  methotrexate.	  Anti-­‐cancer	  drugs	  2005;16:733-­‐8.	  


	  
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	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
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28.	  
Treon	  SP,	  Chabner	  BA.	  Concepts	  in	  use	  of	  high-­‐dose	  methotrexate	  therapy.	  Clinical	  chemistry	  1996;42:1322-­‐
9.	  
29.	  
Mikkelsen	  TS,	  Sparreboom	  A,	  Cheng	  C,	  et	  al.	  Shortening	  infusion	  time	  for	  high-­‐dose	  methotrexate	  alters	  
antileukemic	  effects:	  a	  randomized	  prospective	  clinical	  trial.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  
the	  American	  Society	  of	  Clinical	  Oncology	  2011;29:1771-­‐8.	  
30.	  
Bleyer	  WA.	  The	  clinical	  pharmacology	  of	  methotrexate:	  new	  applications	  of	  an	  old	  drug.	  Cancer	  1978;41:36-­‐
51.	  
31.	  
Widemann	  BC,	  Adamson	  PC.	  Understanding	  and	  managing	  methotrexate	  nephrotoxicity.	  The	  oncologist	  
2006;11:694-­‐703.	  
32.	  
Widemann	  BC,	  Balis	  FM,	  Kempf-­‐Bielack	  B,	  et	  al.	  High-­‐dose	  methotrexate-­‐induced	  nephrotoxicity	  in	  patients	  
with	  osteosarcoma.	  Cancer	  2004;100:2222-­‐32.	  
33.	  
Fox	  RM.	  Methotrexate	  nephrotoxicity.	  Clinical	  and	  experimental	  pharmacology	  &	  physiology	  1979:43-­‐5.	  
34.	  
Ferrari	  S,	  Palmerini	  E,	  Staals	  E,	  et	  al.	  Sex-­‐	  and	  age-­‐related	  chemotherapy	  toxicity	  in	  patients	  with	  non-­‐
metastatic	  osteosarcoma.	  Journal	  of	  chemotherapy	  2009;21:205-­‐10.	  
35.	  
Hempel	  L,	  Misselwitz	  J,	  Fleck	  C,	  et	  al.	  Influence	  of	  high-­‐dose	  methotrexate	  therapy	  (HD-­‐MTX)	  on	  glomerular	  
and	  tubular	  kidney	  function.	  Medical	  and	  pediatric	  oncology	  2003;40:348-­‐54.	  
36.	  
Delepine	  N,	  Delepine	  G,	  Cornille	  H,	  Brion	  F,	  Arnaud	  P,	  Desbois	  JC.	  Dose	  escalation	  with	  pharmacokinetics	  
monitoring	  in	  methotrexate	  chemotherapy	  of	  osteosarcoma.	  Anticancer	  research	  1995;15:489-­‐94.	  
37.	  
Ferrari	  S,	  Sassoli	  V,	  Orlandi	  M,	  et	  al.	  Serum	  methotrexate	  (MTX)	  concentrations	  and	  prognosis	  in	  patients	  
with	  osteosarcoma	  of	  the	  extremities	  treated	  with	  a	  multidrug	  neoadjuvant	  regimen.	  Journal	  of	  
chemotherapy	  1993;5:135-­‐41.	  
38.	  
Bacci	  G,	  Loro	  L,	  Longhi	  A,	  et	  al.	  No	  correlation	  between	  methotrexate	  serum	  level	  and	  histologic	  response	  in	  
the	  pre-­‐operative	  treatment	  of	  extremity	  osteosarcoma.	  Anti-­‐cancer	  drugs	  2006;17:411-­‐5.	  
39.	  
Leung	  S,	  Marshall	  GM,	  al	  Mahr	  M,	  Tobias	  V,	  Lee	  DB,	  Hughes	  DO.	  Prognostic	  significance	  of	  chemotherapy	  
dosage	  characteristics	  in	  children	  with	  osteogenic	  sarcoma.	  Medical	  and	  pediatric	  oncology	  1997;28:179-­‐82.	  
40.	  
Zelcer	  S,	  Kellick	  M,	  Wexler	  LH,	  et	  al.	  Methotrexate	  levels	  and	  outcome	  in	  osteosarcoma.	  Pediatric	  blood	  &	  
cancer	  2005;44:638-­‐42.	  
41.	  
Crews	  KR,	  Liu	  T,	  Rodriguez-­‐Galindo	  C,	  et	  al.	  High-­‐dose	  methotrexate	  pharmacokinetics	  and	  outcome	  of	  
children	  and	  young	  adults	  with	  osteosarcoma.	  Cancer	  2004;100:1724-­‐33.	  
42.	  
Krailo	  M,	  Ertel	  I,	  Makley	  J,	  et	  al.	  A	  randomized	  study	  comparing	  high-­‐dose	  methotrexate	  with	  moderate-­‐
dose	  methotrexate	  as	  components	  of	  adjuvant	  chemotherapy	  in	  childhood	  nonmetastatic	  osteosarcoma:	  a	  
report	  from	  the	  Childrens	  Cancer	  Study	  Group.	  Medical	  and	  pediatric	  oncology	  1987;15:69-­‐77.	  
43.	  
Wang	  D,	  Lippard	  SJ.	  Cellular	  processing	  of	  platinum	  anticancer	  drugs.	  Nature	  reviews	  Drug	  discovery	  
2005;4:307-­‐20.	  
44.	  
Pabla	  N,	  Dong	  Z.	  Cisplatin	  nephrotoxicity:	  mechanisms	  and	  renoprotective	  strategies.	  Kidney	  international	  
2008;73:994-­‐1007.	  
45.	  
Skinner	  R,	  Pearson	  AD,	  English	  MW,	  et	  al.	  Cisplatin	  dose	  rate	  as	  a	  risk	  factor	  for	  nephrotoxicity	  in	  children.	  
British	  journal	  of	  cancer	  1998;77:1677-­‐82.	  
46.	  
Womer	  RB,	  Pritchard	  J,	  Barratt	  TM.	  Renal	  toxicity	  of	  cisplatin	  in	  children.	  The	  Journal	  of	  pediatrics	  
1985;106:659-­‐63.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
39  
47.	  
Launay-­‐Vacher	  V,	  Rey	  JB,	  Isnard-­‐Bagnis	  C,	  Deray	  G,	  Daouphars	  M.	  Prevention	  of	  cisplatin	  nephrotoxicity:	  
state	  of	  the	  art	  and	  recommendations	  from	  the	  European	  Society	  of	  Clinical	  Pharmacy	  Special	  Interest	  
Group	  on	  Cancer	  Care.	  Cancer	  chemotherapy	  and	  pharmacology	  2008;61:903-­‐9.	  
48.	  
Ciarimboli	  G.	  Role	  of	  organic	  cation	  transporters	  in	  drug-­‐induced	  toxicity.	  Expert	  opinion	  on	  drug	  
metabolism	  &	  toxicology	  2011;7:159-­‐74.	  
49.	  
Yokoo	  S,	  Yonezawa	  A,	  Masuda	  S,	  Fukatsu	  A,	  Katsura	  T,	  Inui	  K.	  Differential	  contribution	  of	  organic	  cation	  
transporters,	  OCT2	  and	  MATE1,	  in	  platinum	  agent-­‐induced	  nephrotoxicity.	  Biochemical	  pharmacology	  
2007;74:477-­‐87.	  
50.	  
Ciarimboli	  G,	  Deuster	  D,	  Knief	  A,	  et	  al.	  Organic	  cation	  transporter	  2	  mediates	  cisplatin-­‐induced	  oto-­‐	  and	  
nephrotoxicity	  and	  is	  a	  target	  for	  protective	  interventions.	  The	  American	  journal	  of	  pathology	  
2010;176:1169-­‐80.	  
51.	  
Filipski	  KK,	  Mathijssen	  RH,	  Mikkelsen	  TS,	  Schinkel	  AH,	  Sparreboom	  A.	  Contribution	  of	  organic	  cation	  
transporter	  2	  (OCT2)	  to	  cisplatin-­‐induced	  nephrotoxicity.	  Clinical	  pharmacology	  and	  therapeutics	  
2009;86:396-­‐402.	  
52.	  
Franke	  RM,	  Kosloske	  AM,	  Lancaster	  CS,	  et	  al.	  Influence	  of	  Oct1/Oct2-­‐deficiency	  on	  cisplatin-­‐induced	  changes	  
in	  urinary	  N-­‐acetyl-­‐beta-­‐D-­‐glucosaminidase.	  Clinical	  cancer	  research	  :	  an	  official	  journal	  of	  the	  American	  
Association	  for	  Cancer	  Research	  2010;16:4198-­‐206.	  
53.	  
Wilson	  CL,	  Ness	  KK,	  Neglia	  JP,	  et	  al.	  Renal	  carcinoma	  following	  therapy	  for	  cancer	  in	  childhood:	  A	  report	  
from	  the	  Childhood	  Cancer	  Survivor	  Study	  (Abstr#9528).	  In:	  American	  Society	  of	  Clinical	  Oncology;	  2012;	  
Chicago.	  IL;	  2012.	  p.	  613a.	  
54.	  
Erdlenbruch	  B,	  Nier	  M,	  Kern	  W,	  Hiddemann	  W,	  Pekrun	  A,	  Lakomek	  M.	  Pharmacokinetics	  of	  cisplatin	  and	  
relation	  to	  nephrotoxicity	  in	  paediatric	  patients.	  European	  journal	  of	  clinical	  pharmacology	  2001;57:393-­‐
402.	  
55.	  
Peng	  B,	  English	  MW,	  Boddy	  AV,	  et	  al.	  Cisplatin	  pharmacokinetics	  in	  children	  with	  cancer.	  European	  journal	  
of	  cancer	  1997;33:1823-­‐8.	  
56.	  
Ribrag	  V,	  Droz	  JP,	  Morizet	  J,	  Leclercq	  B,	  Gouyette	  A,	  Chabot	  GG.	  Test	  dose-­‐guided	  administration	  of	  cisplatin	  
in	  an	  anephric	  patient:	  a	  case	  report.	  Annals	  of	  oncology	  :	  official	  journal	  of	  the	  European	  Society	  for	  
Medical	  Oncology	  /	  ESMO	  1993;4:679-­‐82.	  
57.	  
Stevens	  LA,	  Coresh	  J,	  Greene	  T,	  Levey	  AS.	  Assessing	  kidney	  function-­‐-­‐measured	  and	  estimated	  glomerular	  
filtration	  rate.	  The	  New	  England	  journal	  of	  medicine	  2006;354:2473-­‐83.	  
58.	  
Schwartz	  GJ,	  Munoz	  A,	  Schneider	  MF,	  et	  al.	  New	  equations	  to	  estimate	  GFR	  in	  children	  with	  CKD.	  Journal	  of	  
the	  American	  Society	  of	  Nephrology	  :	  JASN	  2009;20:629-­‐37.	  
59.	  
Conti	  M,	  Moutereau	  S,	  Zater	  M,	  et	  al.	  Urinary	  cystatin	  C	  as	  a	  specific	  marker	  of	  tubular	  dysfunction.	  Clinical	  
chemistry	  and	  laboratory	  medicine	  :	  CCLM	  /	  FESCC	  2006;44:288-­‐91.	  
60.	  
Cordeiro	  VF,	  Pinheiro	  DC,	  Silva	  GB,	  Jr.,	  et	  al.	  Comparative	  study	  of	  cystatin	  C	  and	  serum	  creatinine	  in	  the	  
estimative	  of	  glomerular	  filtration	  rate	  in	  children.	  Clinica	  chimica	  acta;	  international	  journal	  of	  clinical	  
chemistry	  2008;391:46-­‐50.	  
61.	  
Dharnidharka	  VR,	  Kwon	  C,	  Stevens	  G.	  Serum	  cystatin	  C	  is	  superior	  to	  serum	  creatinine	  as	  a	  marker	  of	  kidney	  
function:	  a	  meta-­‐analysis.	  American	  journal	  of	  kidney	  diseases	  :	  the	  official	  journal	  of	  the	  National	  Kidney	  
Foundation	  2002;40:221-­‐6.	  
62.	  
Gronroos	  MH,	  Jahnukainen	  T,	  Irjala	  K,	  et	  al.	  Comparison	  of	  glomerular	  function	  tests	  in	  children	  with	  cancer.	  
Pediatric	  nephrology	  2008;23:797-­‐803.	  
63.	  
Grubb	  AO.	  Cystatin	  C-­‐-­‐properties	  and	  use	  as	  diagnostic	  marker.	  Advances	  in	  clinical	  chemistry	  2000;35:63-­‐
99.	  


	  
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	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
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64.	  
Herget-­‐Rosenthal	  S,	  Feldkamp	  T,	  Volbracht	  L,	  Kribben	  A.	  Measurement	  of	  urinary	  cystatin	  C	  by	  particle-­‐
enhanced	  nephelometric	  immunoassay:	  precision,	  interferences,	  stability	  and	  reference	  range.	  Annals	  of	  
clinical	  biochemistry	  2004;41:111-­‐8.	  
65.	  
Herget-­‐Rosenthal	  S,	  Marggraf	  G,	  Husing	  J,	  et	  al.	  Early	  detection	  of	  acute	  renal	  failure	  by	  serum	  cystatin	  C.	  
Kidney	  international	  2004;66:1115-­‐22.	  
66.	  
Roos	  JF,	  Doust	  J,	  Tett	  SE,	  Kirkpatrick	  CM.	  Diagnostic	  accuracy	  of	  cystatin	  C	  compared	  to	  serum	  creatinine	  for	  
the	  estimation	  of	  renal	  dysfunction	  in	  adults	  and	  children-­‐-­‐a	  meta-­‐analysis.	  Clinical	  biochemistry	  
2007;40:383-­‐91.	  
67.	  
Westhuyzen	  J.	  Cystatin	  C:	  a	  promising	  marker	  and	  predictor	  of	  impaired	  renal	  function.	  Annals	  of	  clinical	  
and	  laboratory	  science	  2006;36:387-­‐94.	  
68.	  
Zaffanello	  M,	  Franchini	  M,	  Fanos	  V.	  Is	  serum	  Cystatin-­‐C	  a	  suitable	  marker	  of	  renal	  function	  in	  children?	  
Annals	  of	  clinical	  and	  laboratory	  science	  2007;37:233-­‐40.	  
69.	  
Du	  Y,	  Zappitelli	  M,	  Mian	  A,	  et	  al.	  Urinary	  biomarkers	  to	  detect	  acute	  kidney	  injury	  in	  the	  pediatric	  emergency	  
center.	  Pediatric	  nephrology	  2011;26:267-­‐74.	  
70.	  
Han	  WK,	  Bailly	  V,	  Abichandani	  R,	  Thadhani	  R,	  Bonventre	  JV.	  Kidney	  Injury	  Molecule-­‐1	  (KIM-­‐1):	  a	  novel	  
biomarker	  for	  human	  renal	  proximal	  tubule	  injury.	  Kidney	  international	  2002;62:237-­‐44.	  
71.	  
Han	  WK,	  Wagener	  G,	  Zhu	  Y,	  Wang	  S,	  Lee	  HT.	  Urinary	  biomarkers	  in	  the	  early	  detection	  of	  acute	  kidney	  injury	  
after	  cardiac	  surgery.	  Clinical	  journal	  of	  the	  American	  Society	  of	  Nephrology	  :	  CJASN	  2009;4:873-­‐82.	  
72.	  
Koyner	  JL,	  Vaidya	  VS,	  Bennett	  MR,	  et	  al.	  Urinary	  biomarkers	  in	  the	  clinical	  prognosis	  and	  early	  detection	  of	  
acute	  kidney	  injury.	  Clinical	  journal	  of	  the	  American	  Society	  of	  Nephrology	  :	  CJASN	  2010;5:2154-­‐65.	  
73.	  
Liangos	  O,	  Perianayagam	  MC,	  Vaidya	  VS,	  et	  al.	  Urinary	  N-­‐acetyl-­‐beta-­‐(D)-­‐glucosaminidase	  activity	  and	  
kidney	  injury	  molecule-­‐1	  level	  are	  associated	  with	  adverse	  outcomes	  in	  acute	  renal	  failure.	  Journal	  of	  the	  
American	  Society	  of	  Nephrology	  :	  JASN	  2007;18:904-­‐12.	  
74.	  
de	  Geus	  HR,	  Bakker	  J,	  Lesaffre	  EM,	  le	  Noble	  JL.	  Neutrophil	  gelatinase-­‐associated	  lipocalin	  at	  ICU	  admission	  
predicts	  for	  acute	  kidney	  injury	  in	  adult	  patients.	  American	  journal	  of	  respiratory	  and	  critical	  care	  medicine	  
2011;183:907-­‐14.	  
75.	  
Dent	  CL,	  Ma	  Q,	  Dastrala	  S,	  et	  al.	  Plasma	  neutrophil	  gelatinase-­‐associated	  lipocalin	  predicts	  acute	  kidney	  
injury,	  morbidity	  and	  mortality	  after	  pediatric	  cardiac	  surgery:	  a	  prospective	  uncontrolled	  cohort	  study.	  
Critical	  care	  2007;11:R127.	  
76.	  
Haase	  M,	  Bellomo	  R,	  Devarajan	  P,	  Schlattmann	  P,	  Haase-­‐Fielitz	  A.	  Accuracy	  of	  neutrophil	  gelatinase-­‐
associated	  lipocalin	  (NGAL)	  in	  diagnosis	  and	  prognosis	  in	  acute	  kidney	  injury:	  a	  systematic	  review	  and	  meta-­‐
analysis.	  American	  journal	  of	  kidney	  diseases	  :	  the	  official	  journal	  of	  the	  National	  Kidney	  Foundation	  
2009;54:1012-­‐24.	  
77.	  
Haase	  M,	  Bellomo	  R,	  Haase-­‐Fielitz	  A.	  Neutrophil	  gelatinase-­‐associated	  lipocalin:	  a	  superior	  biomarker	  for	  
detection	  of	  subclinical	  acute	  kidney	  injury	  and	  poor	  prognosis.	  Biomarkers	  in	  medicine	  2011;5:415-­‐7.	  
78.	  
Haase	  M,	  Devarajan	  P,	  Haase-­‐Fielitz	  A,	  et	  al.	  The	  outcome	  of	  neutrophil	  gelatinase-­‐associated	  lipocalin-­‐
positive	  subclinical	  acute	  kidney	  injury:	  a	  multicenter	  pooled	  analysis	  of	  prospective	  studies.	  Journal	  of	  the	  
American	  College	  of	  Cardiology	  2011;57:1752-­‐61.	  
79.	  
Nickolas	  TL,	  O'Rourke	  MJ,	  Yang	  J,	  et	  al.	  Sensitivity	  and	  specificity	  of	  a	  single	  emergency	  department	  
measurement	  of	  urinary	  neutrophil	  gelatinase-­‐associated	  lipocalin	  for	  diagnosing	  acute	  kidney	  injury.	  
Annals	  of	  internal	  medicine	  2008;148:810-­‐9.	  
80.	  
Schmidt-­‐Ott	  KM,	  Mori	  K,	  Li	  JY,	  et	  al.	  Dual	  action	  of	  neutrophil	  gelatinase-­‐associated	  lipocalin.	  Journal	  of	  the	  
American	  Society	  of	  Nephrology	  :	  JASN	  2007;18:407-­‐13.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
41  
81.	  
Shemin	  D,	  Dworkin	  LD.	  Neutrophil	  gelatinase-­‐associated	  lipocalin	  (NGAL)	  as	  a	  biomarker	  for	  early	  acute	  
kidney	  injury.	  Critical	  care	  clinics	  2011;27:379-­‐89.	  
82.	  
Singer	  E,	  Elger	  A,	  Elitok	  S,	  et	  al.	  Urinary	  neutrophil	  gelatinase-­‐associated	  lipocalin	  distinguishes	  pre-­‐renal	  
from	  intrinsic	  renal	  failure	  and	  predicts	  outcomes.	  Kidney	  international	  2011;80:405-­‐14.	  
83.	  
Sise	  ME,	  Forster	  C,	  Singer	  E,	  et	  al.	  Urine	  neutrophil	  gelatinase-­‐associated	  lipocalin	  identifies	  unilateral	  and	  
bilateral	  urinary	  tract	  obstruction.	  Nephrology,	  dialysis,	  transplantation	  :	  official	  publication	  of	  the	  European	  
Dialysis	  and	  Transplant	  Association	  -­‐	  European	  Renal	  Association	  2011;26:4132-­‐5.	  
84.	  
Tuladhar	  SM,	  Puntmann	  VO,	  Soni	  M,	  Punjabi	  PP,	  Bogle	  RG.	  Rapid	  detection	  of	  acute	  kidney	  injury	  by	  plasma	  
and	  urinary	  neutrophil	  gelatinase-­‐associated	  lipocalin	  after	  cardiopulmonary	  bypass.	  Journal	  of	  
cardiovascular	  pharmacology	  2009;53:261-­‐6.	  
85.	  
Wheeler	  DS,	  Devarajan	  P,	  Ma	  Q,	  et	  al.	  Serum	  neutrophil	  gelatinase-­‐associated	  lipocalin	  (NGAL)	  as	  a	  marker	  
of	  acute	  kidney	  injury	  in	  critically	  ill	  children	  with	  septic	  shock.	  Critical	  care	  medicine	  2008;36:1297-­‐303.	  
86.	  
Waring	  WS,	  Moonie	  A.	  Earlier	  recognition	  of	  nephrotoxicity	  using	  novel	  biomarkers	  of	  acute	  kidney	  injury.	  
Clinical	  toxicology	  2011;49:720-­‐8.	  
87.	  
Brock	  PR,	  Knight	  KR,	  Freyer	  DR,	  et	  al.	  Platinum-­‐induced	  ototoxicity	  in	  children:	  a	  consensus	  review	  on	  
mechanisms,	  predisposition,	  and	  protection,	  including	  a	  new	  international	  society	  of	  pediatric	  oncology	  
Boston	  ototoxicity	  scale.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  
Oncology	  2012;30:2408-­‐17.	  
88.	  
Lewis	  MJ,	  DuBois	  SG,	  Fligor	  B,	  Li	  X,	  Goorin	  A,	  Grier	  HE.	  Ototoxicity	  in	  children	  treated	  for	  osteosarcoma.	  
Pediatric	  blood	  &	  cancer	  2009;52:387-­‐91.	  
89.	  
Ruiz	  L,	  Gilden	  J,	  Jaffe	  N,	  Robertson	  R,	  Wang	  YM.	  Auditory	  function	  in	  pediatric	  osteosarcoma	  patients	  
treated	  with	  multiple	  doses	  of	  cis-­‐diamminedichloroplatinum(II).	  Cancer	  research	  1989;49:742-­‐4.	  
90.	  
Stohr	  W,	  Langer	  T,	  Kremers	  A,	  et	  al.	  Cisplatin-­‐induced	  ototoxicity	  in	  osteosarcoma	  patients:	  a	  report	  from	  
the	  late	  effects	  surveillance	  system.	  Cancer	  investigation	  2005;23:201-­‐7.	  
91.	  
Li	  Y,	  Womer	  RB,	  Silber	  JH.	  Predicting	  cisplatin	  ototoxicity	  in	  children:	  the	  influence	  of	  age	  and	  the	  cumulative	  
dose.	  European	  journal	  of	  cancer	  2004;40:2445-­‐51.	  
92.	  
Yancey	  A,	  Harris	  MS,	  Egbelakin	  A,	  Gilbert	  J,	  Pisoni	  DB,	  Renbarger	  J.	  Risk	  factors	  for	  cisplatin-­‐associated	  
ototoxicity	  in	  pediatric	  oncology	  patients.	  Pediatric	  blood	  &	  cancer	  2012.	  
93.	  
Bertolini	  P,	  Lassalle	  M,	  Mercier	  G,	  et	  al.	  Platinum	  compound-­‐related	  ototoxicity	  in	  children:	  long-­‐term	  
follow-­‐up	  reveals	  continuous	  worsening	  of	  hearing	  loss.	  Journal	  of	  pediatric	  hematology/oncology	  
2004;26:649-­‐55.	  
94.	  
Moreira	  Dias	  L.	  Pantoprazole:	  a	  proton	  pump	  inhibitor.	  Clinical	  drug	  investigation	  2009;29	  Suppl	  2:3-­‐12.	  
95.	  
Katsuda	  H,	  Yamashita	  M,	  Katsura	  H,	  et	  al.	  Protecting	  cisplatin-­‐induced	  nephrotoxicity	  with	  cimetidine	  does	  
not	  affect	  antitumor	  activity.	  Biological	  &	  pharmaceutical	  bulletin	  2010;33:1867-­‐71.	  
96.	  
De	  Milito	  A,	  Fais	  S.	  Proton	  pump	  inhibitors	  may	  reduce	  tumour	  resistance.	  Expert	  Opin	  Pharmacother	  
2005;6:1049-­‐54.	  
97.	  
Luciani	  F,	  Spada	  M,	  De	  Milito	  A,	  et	  al.	  Effect	  of	  proton	  pump	  inhibitor	  pretreatment	  on	  resistance	  of	  solid	  
tumors	  to	  cytotoxic	  drugs.	  J	  Natl	  Cancer	  Inst	  2004;96:1702-­‐13.	  
98.	  
Suzuki	  K,	  Doki	  K,	  Homma	  M,	  et	  al.	  Co-­‐administration	  of	  proton	  pump	  inhibitors	  delays	  elimination	  of	  plasma	  
methotrexate	  in	  high-­‐dose	  methotrexate	  therapy.	  British	  journal	  of	  clinical	  pharmacology	  2009;67:44-­‐9.	  
99.	  
Bacci	  G,	  Longhi	  A,	  Versari	  M,	  Mercuri	  M,	  Briccoli	  A,	  Picci	  P.	  Prognostic	  factors	  for	  osteosarcoma	  of	  the	  
extremity	  treated	  with	  neoadjuvant	  chemotherapy:	  15-­‐year	  experience	  in	  789	  patients	  treated	  at	  a	  single	  
institution.	  Cancer	  2006;106:1154-­‐61.	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
42  
100.	   Leung	  KS,	  Fung	  KP,	  Sher	  AH,	  Li	  CK,	  Lee	  KM.	  Plasma	  bone-­‐specific	  alkaline	  phosphatase	  as	  an	  indicator	  of	  
osteoblastic	  activity.	  The	  Journal	  of	  bone	  and	  joint	  surgery	  British	  volume	  1993;75:288-­‐92.	  
101.	   Rauch	  F,	  Middelmann	  B,	  Cagnoli	  M,	  Keller	  KM,	  Schonau	  E.	  Comparison	  of	  total	  alkaline	  phosphatase	  and	  
three	  assays	  for	  bone-­‐specific	  alkaline	  phosphatase	  in	  childhood	  and	  adolescence.	  Acta	  paediatrica	  
1997;86:583-­‐7.	  
102.	   Wang	  J,	  Pei	  F,	  Tu	  C,	  Zhang	  H,	  Qiu	  X.	  Serum	  bone	  turnover	  markers	  in	  patients	  with	  primary	  bone	  tumors.	  
Oncology	  2007;72:338-­‐42.	  
103.	   Limmahakhun	  S,	  Pothacharoen	  P,	  Theera-­‐Umpon	  N,	  et	  al.	  Relationships	  between	  serum	  biomarker	  levels	  
and	  clinical	  presentation	  of	  human	  osteosarcomas.	  Asian	  Pacific	  journal	  of	  cancer	  prevention	  :	  APJCP	  
2011;12:1717-­‐22.	  
104.	   Morgan	  KP,	  Buie	  LW,	  Savage	  SW.	  The	  role	  of	  mannitol	  as	  a	  nephroprotectant	  in	  patients	  receiving	  cisplatin	  
therapy.	  The	  Annals	  of	  pharmacotherapy	  2012;46:276-­‐81.	  
105.	   Santoso	  JT,	  Lucci	  JA,	  3rd,	  Coleman	  RL,	  Schafer	  I,	  Hannigan	  EV.	  Saline,	  mannitol,	  and	  furosemide	  hydration	  in	  
acute	  cisplatin	  nephrotoxicity:	  a	  randomized	  trial.	  Cancer	  chemotherapy	  and	  pharmacology	  2003;52:13-­‐8.	  
106.	   DeLaney	  TF,	  Park	  L,	  Goldberg	  SI,	  et	  al.	  Radiotherapy	  for	  local	  control	  of	  osteosarcoma.	  International	  journal	  
of	  radiation	  oncology,	  biology,	  physics	  2005;61:492-­‐8.	  
107.	   Schwarz	  R,	  Bruland	  O,	  Cassoni	  A,	  Schomberg	  P,	  Bielack	  S.	  The	  role	  of	  radiotherapy	  in	  oseosarcoma.	  Cancer	  
treatment	  and	  research	  2009;152:147-­‐64.	  
108.	   Harris	  MB,	  Gieser	  P,	  Goorin	  AM,	  et	  al.	  Treatment	  of	  metastatic	  osteosarcoma	  at	  diagnosis:	  a	  Pediatric	  
Oncology	  Group	  Study.	  Journal	  of	  clinical	  oncology	  :	  official	  journal	  of	  the	  American	  Society	  of	  Clinical	  
Oncology	  1998;16:3641-­‐8.	  
109.	   Karrison	  TG,	  Maitland	  ML,	  Stadler	  WM,	  Ratain	  MJ.	  Design	  of	  phase	  II	  cancer	  trials	  using	  a	  continuous	  
endpoint	  of	  change	  in	  tumor	  size:	  application	  to	  a	  study	  of	  sorafenib	  and	  erlotinib	  in	  non	  small-­‐cell	  lung	  
cancer.	  J	  Natl	  Cancer	  Inst	  2007;99:1455-­‐61.	  
110.	   Muller	  KE,	  Peterson	  BL.	  Practical	  methods	  for	  computing	  power	  in	  testing	  the	  multivariate	  general	  linear	  
hypothesis.	  Computat	  Stat	  Data	  Analysis	  1984;2:143-­‐58.	  
111.	   Muller	  KE,	  LaVange	  LM,	  Ramey	  SL,	  Ramey	  CT.	  Power	  calculations	  for	  general	  linear	  multivariate	  models	  
including	  repeated	  measures	  applications.	  J	  Am	  Stat	  Assoc	  1992;87:1209-­‐26.	  
112.	   Chaturvedi	  S,	  Farmer	  T,	  Kapke	  GF.	  Assay	  validation	  for	  KIM-­‐1:	  human	  urinary	  renal	  dysfunction	  biomarker.	  
International	  journal	  of	  biological	  sciences	  2009;5:128-­‐34.	  
113.	   McCarthy	  DM.	  Proton	  pump	  inhibitor	  use	  and	  Clostridium	  difficile	  colitis:	  cause	  or	  coincidence?	  Journal	  of	  
clinical	  gastroenterology	  2012;46:350-­‐3.	  
114.	   Deshpande	  A,	  Pant	  C,	  Pasupuleti	  V,	  et	  al.	  Association	  between	  proton	  pump	  inhibitor	  therapy	  and	  
Clostridium	  difficile	  infection	  in	  a	  meta-­‐analysis.	  Clinical	  gastroenterology	  and	  hepatology	  :	  the	  official	  
clinical	  practice	  journal	  of	  the	  American	  Gastroenterological	  Association	  2012;10:225-­‐33.	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
43  
APPENDIX	  1	   SAMPLE	  TREATMENT	  DELIVERY	  MAP	  
Protocol'No.
Name
Version'Date
MRN
DOB
C1/D1
C1/D22
C2/D1
C2/D22
Cycles 1'and'2
Weight'(kg)
Treatment'Arm 1
Height'(cm)
BSA'(m2)
Drug
Dose
Route
Schedule
Cycle
Cycle4Days
Dexrazoxane
375'mg/m2
IV
over'15'min
1'and'2
1'and'2
DOXOrubicin
37.5'mg/m2
IV
over'15'min
1'and'2
1'and'2
0.3'mg/kg
IV
over'15'min
1'and'2
1'and'2
1.3'mg/kg
IV
over'4'h
1'and'2
1'and'2
CISplatin
60'mg/m2
IV
over'4'h
1'and'2
1'and'2
Pegfilgrastim
0.1'mg/kg
SC
1'and'2
3
Methotrexate
12'gm/m2
IV
over'4'h
1'and'2
22'and'29
Leucovorin
15'mg/m2
IV'or'
PO
q6h'until'
[MTX]<0.1mcM
1'and'2
23'and'30
Date
Cycle
Day
%4Dose*
Dose
Units
1
1
mg
mg
mg
mg
mg
1
2
mg
mg
mg
mg
mg
1
3
mg
1
22
Methotrexate
over'4'h
mg
23
Leucovorin
mg
1
29
Methotrexate
over'4'h
mg
30
Leucovorin
mg
2
1
mg
mg
mg
mg
mg
2
2
mg
mg
mg
mg
mg
1
3
mg
2
22
Methotrexate
over'4'h
mg
23
Leucovorin
mg
2
29
Methotrexate
over'4'h
mg
30
Leucovorin
mg
*'%'of'standard'dose.'100%'is'full'dose;'75%'is'a'25%'dose'reduction.
DOXOrubicin
Pantoprazole'(loading)
Pantoprazole'(infusion)
CISplatin
Pegfilgrastim
0
0
0
Dexrazoxane
DOXOrubicin
Pantoprazole'(loading)
Pantoprazole'(infusion)
CISplatin
Dexrazoxane
Dexrazoxane
DOXOrubicin
Pantoprazole'(loading)
Pantoprazole'(infusion)
CISplatin
Pantoprazole'(loading)
Pantoprazole'(infusion)
CISplatin
Pegfilgrastim
Comment
MAX'20'gm
leucovorin'must'start'24'h'after'the'
start'of'the'MTX'infusion
Drug
Dexrazoxane
DOXOrubicin
MAX'6'mg
June'13,'2012
Pantoprazole
TREATMENT4DELIVERY4MAP
CHP12STXX
Comment
infuse'prior'to'DOXOrubicin
infuse'prior'to'cisplatin
infuse'with'cisplatin
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
44  
	  
SAMPLE	  CORRELATIVE	  STUDIES	  MAP	  
	  
Protocol'No.
Name
Version'Date
MRN
DOB
Date
Time
Cycle
Day
Specimen
Test
Comment
1
1
Urine
AKI,'Cr
Urine
Mg,'Cr
Plasma
Mg,'Cr
Serum
Cystatin'C
1
2
Urine
Mg,'Cr
Plasma
Mg,'Cr
Urine
AKI,'Cr
1
7
Urine
AKI,'Cr
Urine
Mg,'Cr
Plasma
Mg,'Cr
Serum
Cystatin'C
1
22
Urine
AKI,'Cr
Serum
Cystatin'C
Urine
MTX
1
23
Urine
AKI,'Cr
1
29
Urine
AKI,'Cr
Serum
Cystatin'C
Urine
MTX
1
30
Urine
AKI,'Cr
2
1
Urine
AKI,'Cr
Urine
Mg,'Cr
Plasma
Mg,'Cr
Serum
Cystatin'C
Plasma
PreHalbumin
H
PRO
H
Arm'circ/'
Skin'fold
2
2
Urine
Mg,'Cr
Plasma
Mg,'Cr
Urine
AKI,'Cr
2
7
Urine
AKI,'Cr
Urine
Mg,'Cr
Plasma
Mg,'Cr
Serum
Cystatin'C
2
22
Urine
AKI,'Cr
Serum
Cystatin'C
Urine
MTX
2
23
Urine
AKI,'Cr
2
29
Urine
AKI,'Cr
Serum
Cystatin'C
Urine
MTX
2
30
Urine
AKI,'Cr
Prior'to'D1'cisplatin'infusion
Prior'to'D2'cisplatin'infusion
Prior'to'D22'HDMTX'infusion
End'of'D22'HDMTX'infusion
24'h'after'start'of'D22'HDMTX'infusion
Instructions
End'of'D2'cisplatin'infusion
End'of'D22'HDMTX'infusion
24'h'after'start'of'D22'HDMTX'infusion
Prior'to'D29'HDMTX'infusion
End'of'D29'HDMTX'infusion
24'h'after'start'of'D29''HDMTX'infusion
Prior'to'D29'HDMTX'infusion
End'of'D29'HDMTX'infusion
24'h'after'start'of'D29''HDMTX'infusion
Prior'to'D2'cisplatin'infusion
Prior'to'D22'HDMTX'infusion
Prior'to'D1'cisplatin'infusion
End'of'D2'cisplatin'infusion
CHP12ST051
,'
January'10,'2013
0
1/0/1900
CORRELATIVE:STUDIES:MAP:>:CYCLES:1:AND:2


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
45  
APPENDIX	  2	   PEDIATRIC	  NEUROTOXICITY	  GRADING	  SCALE	  
Motor	  neuropathy:	  
• Grade	  1:	   Subjective	  weakness,	  but	  no	  deficits	  detected	  on	  neurological	  exam	  
• Grade	  2:	   Weakness	  that	  alters	  fine	  motor	  skills	  (buttoning	  shirt,	  writing	  or	  drawing,	  
using	  eating	  utensils)	  or	  gait	  without	  abrogating	  ability	  to	  perform	  these	  tasks.	  
• Grade	  3:	   Unable	  to	  perform	  fine	  motor	  tasks	  (buttoning	  shirt,	  writing	  or	  drawing,	  
using	  eating	  utensils)	  or	  unable	  to	  ambulate	  without	  assistance.	  
• Grade	  4:	   Paralysis	  
Sensory	  neuropathy	  :	  
• Grade	  1:	   Paresthesias,	  pain,	  or	  numbness	  that	  do	  not	  require	  treatment	  or	  interfere	  
with	  extremity	  function.	  
• Grade	  2:	   Paresthesias,	  pain,	  or	  numbness	  that	  is	  controlled	  by	  non-­‐narcotic	  
medications,	  or	  alter	  (without	  causing	  loss	  of	  function)	  fine	  motor	  skills	  (buttoning	  shirt,	  
writing	  or	  drawing,	  using	  eating	  utensils)	  or	  gait,	  without	  abrogating	  ability	  to	  perform	  
these	  tasks.	  
• Grade	  3:	   Paresthesias	  or	  pain	  that	  is	  controlled	  by	  narcotics,	  or	  interfere	  with	  
extremity	  function	  (gait,	  fine	  motor	  skills	  as	  outlined	  above),	  or	  quality	  of	  life	  (loss	  of	  
sleep,	  ability	  to	  perform	  normal	  activities	  severely	  impaired).	  
• Grade	  4:	   Complete	  loss	  of	  sensation	  or	  pain	  that	  is	  not	  controlled	  by	  narcotics	  
	  
	  
	  


	  
Protocol	  number:	   CHP12ST051	  
	  Amendment	  number:	   	  
	  
Version	  date:	   April	  2,	  2013	  
	  
	  
46  
APPENDIX	  3	   PATIENT	  REPORTED	  OUTCOME	  SURVEY	  
OSTEOSARCOMA	  PATIENT	  SURVEY	  	  	  
PATIENT	  ID____________	  
	  
	  
DATE__________	  
	  
Since	  the	  last	  time	  I	  filled	  this	  form	  out	  .	  .	  .	  
Never	  
(0	  times)	  
Rarely	  
(less	  than	  
once	  a	  week)	  
Sometimes	  
	  (1	  to	  3	  times	  
a	  week)	  
Often	  
(almost	  
daily)	  
Almost	  
Always	  
(daily)	  
I	  feel	  too	  tired	  to	  get	  out	  of	  bed	  
	  
	  
	  
	  
	  
I	  feel	  like	  I’m	  going	  to	  throw	  up	  
	  
	  
	  
	  
	  
I	  throw-­‐up	  or	  vomit	  
	  
	  
	  
	  
	  
My	  stomach	  hurts	  
	  
	  
	  
	  
	  
I	  do	  not	  feel	  as	  hungry	  as	  I	  use	  to	  
	  
	  
	  
	  
	  
Foods	  taste	  different	  or	  funny	  
	  
	  
	  
	  
	  
I	  have	  diarrhea	  
	  
	  
	  
	  
	  
I	  have	  constipation	  
	  
	  
	  
	  
	  
I	  have	  a	  ringing	  sound	  in	  my	  ears	  
	  
	  
	  
	  
	  
I	  have	  trouble	  hearing	  some	  things	  
	  
	  
	  
	  
	  
My	  mouth	  or	  throat	  hurts	  
	  
	  
	  
	  
	  
I	  am	  in	  pain	  
	  
	  
	  
	  
	  
I	   have	   trouble	   keeping	   up	   with	   school	   work	  
because	   of	   being	   in	   the	   hospital	   or	   feeling	   too	  
sick	  
	  
	  
	  
	  
	  
I	  have	  trouble	  falling	  asleep	  	  
	  
	  
	  
	  
	  
I	  wake	  up	  at	  night	  because	  of	  pain	  
	  
	  
	  
	  
	  
I	  don’t	  see	  my	  friends	  as	  much	  anymore	  because	  
I	  am	  too	  busy	  with	  doctors	  and	  hospital	  visits	  
	  
	  
	  
	  
	  
I	  don’t	  do	  as	  many	  fun	  things	  anymore	  because	  I	  
feel	  tired	  or	  sick	  
	  
	  
	  
	  
	  
I	  don’t	  have	  fun	  doing	  things	  I	  used	  to	  like	  to	  do	  
	  
	  
	  
	  
	  
I	  feel	  nervous,	  worried,	  or	  anxious	  
	  
	  
	  
	  
	  
I	   have	   trouble	   doing	   daily	   activities	   like	   eating,	  
drinking,	  putting	  on	  clothes	  
	  
	  
	  
	  
	  
I	  have	  answered	  these	  questions	  before	  	  (circle	  one)	  
YES	  
NO	  
I	  Don’t	  Know	  
Compared	  to	  the	  last	  time	  I	  answered	  these	  questions,	  I	  feel	  	  	  (circle	  one)	  
	  
BETTER	  
WORSE	  
THE	  SAME	  
I	  DON’T	  KNOW	  
I	  would	  like	  to	  meet	  with	  someone	  at	  the	  hospital	  talk	  about	  how	  I	  feel	  	  (circle	  one)	  
	  
YES	  
NO	  
I	  DON’T	  KNOW	  
I	  AM	  ALREADY	  MEETING	  WITH	  SOMEONE