Clinical trial NCT02076997
Conditions: Cancer
Keywords: Cancer High Dose Methotrexate
Sponsor: Baylor College of Medicine
Start date: 2014-01-01; Completion date: 2019-05-10
Enrollment: 23 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Pediatric cancer patients are being asked to take part in this study who have a cancer that is treated with high doses of the drug methotrexate (MTX). In addition, these patients have either had significant side effects to methotrexate in the past or their doctor thinks that they are at high risk for side effects from receiving methotrexate. Methotrexate is a cancer-fighting drug that is very important in the treatment of leukemia.

In this study, investigators are testing a new method of giving high dose methotrexate to cancer patients which may reduce the chances that the level of methotrexate in the blood is too high. When the levels are too high this is thought to lead to an increase in side effects. Side effects are unintended and unwanted results of treatment.

The initial ordered amount of methotrexate and the period over which methotrexate is given will not change from the current standard of care (meaning what is usually done by doctors, and would likely be done if the patient was not on this study). This study is testing a new method of monitoring and potentially adjusting the final amount of methotrexate that the patient will end up receiving based on levels of methotrexate in the blood in the first 24 hours in order to try to prevent side effects in patients with a previous history of side effects from methotrexate or who are at high risk for having side effects.

On this study the investigators will check methotrexate levels in the blood 2 hours after the patient starts receiving the drug and the investigators will lower the dose of methotrexate if needed. Investigators will do the same thing again 6-8 hours later.

Investigators will also collect an optional blood sample from the patient because the investigators want to study how genetic (DNA) differences are involved in how the body processes methotrexate.

Detailed description:
The study participant will receive the drug methotrexate as a patient in the hospital. High dose methotrexate will be given as a 24-hour infusion as part of the care determined to be necessary by their doctor. Infusion means the drug is given using a needle or tubing inserted into a vein (also called IV).

The standard of care in patients receiving high dose methotrexate is to give the entire dose of methotrexate, without adjustment, and to then check the concentration of methotrexate in the blood at the end of the infusion. Most patients will have a level of methotrexate in their blood which is good to treat cancer and keep side effects at a minimum. However, other patients have a level of methotrexate at the end of the 24-hour infusion which is very high and is thought to lead to an increase in side effects.

In this study, investigators are testing a method of giving methotrexate which may reduce the chances that the level of methotrexate in the blood at the end of the infusion is too high. This will be done by checking the methotrexate level in the blood at two times during the 24-hour infusion and reducing the dose of methotrexate being given if the levels are not thought to be in the correct range (the level is too high). Reducing the dose may result in the patient receiving a lower total dose of methotrexate than initially ordered. However, it may also result in a more preferred concentration of methotrexate in the blood at the end of the infusion.

It is possible that in the past the patient's doctor may have chosen to reduce the amount of methotrexate given because the patient had side effects. In this study, all patients will start out receiving the standard dose of methotrexate. Therefore, by participating in this study, the amount of methotrexate ordered at the start of the infusion may be higher than what the patient received previously.

Before and after the patient receives high dose methotrexate, the patient will receive IV fluids. In order to minimize side effects, methotrexate levels will also be closely monitored after the infusion and the patient will receive supportive medicines as needed. The patient will receive the drug Leucovorin (administered by mouth every 6 hours for minimum of 3 doses). The monitoring and supportive care that the patient will receive at the end of the methotrexate infusion, the amount of IV fluids, and the dose or duration of Leucovorin received will be done based on standard guidelines and are not part of this study.

After receiving the 24-hour high dose methotrexate, this individualized monitoring plan will end and management of the patient's methotrexate levels will continue according to standard guidelines.

High dose methotrexate may be given to the patient multiple times at the discretion of their doctor. While on this study, the patient will receive methotrexate using the methods of this study (however, they will only have blood drawn one time for optional DNA testing). The patient, his/her parent, or the doctor may decide to withdraw the patient from this study at any time if they do not think that it is in the patient's best interest to continue to receive methotrexate in the manner being investigated in this study.

STUDY RELATED TESTS AND PROCEDURES

At study entry, the following will occur:

* The patient will have a physical exam
* Collection of blood samples for routine blood tests and research blood tests. Approximately 1-2 teaspoons of blood will be taken for research blood tests.
* Review of the patient's medical and medication history to collect information.

During treatment and after treatment the following will occur:

* Collection of blood samples for routine blood tests and research blood tests. Approximately ½ teaspoon of blood will be taken at times 2, 6 (or 8), 24, 36, 42, and 48 hours. The blood collected at 2 and 6 (or 8) and 24 hours is for the research blood tests.
* Review of the patient's medical and medication history to collect information.

The research blood tests are done to see how much methotrexate is in the body, how the body handles the drug, and to do DNA studies. The DNA will be tested to look for small changes called Single Nucleotide Polymorphisms (SNP). These small changes sometimes explain why different people have different reactions to drugs. The total amount of blood drawn for the research studies is approximately 3 teaspoons.

Arms: arm 1: Individualized high dose methotrexate given as a 24-hour infusion.
Interventions: intervention 1: Methotrexate
Intervention descriptions: intervention 1: Patients will receive 5g/m\^2 high dose methotrexate as a 24 hour infusion.

The methotrexate level in the blood will be checked at two times during the 24-infusion. The dose will be reduced based on the level in the blood.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Individualized high dose methotrexate for the treatment of malignancies in 
children and adolescents with a significant risk for methotrexate toxicities 
 
 
 
Principal Investigator: 
Jennifer Foster, MD, MPH 
 
 
Co-Investigators: 
Eric S. Schafer, MD, MHS 
Donald “Will” Parsons, MD/PhD 
 
Statistics: 
E. O’Brian Smith, PhD 
 
 
 
 
 
 
 
 
 
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2 
List of Participating Institutions 
 
NO. 
Name of Institution 
1 
Texas Children’s Hospital, Baylor College of Medicine 
2 
University of California San Francisco 
3 
Children’s Hospital of Pennsylvania 
4 
Dana Farber Cancer Institute 
 
 
 
 
 
 


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Table of Contents 
 
Section  
 
 
 
 
 
 
 
 
 
 
 
Page 
 
1. GOALS AND OBJECTIVES  
 
 
 
 
 
 
 
 
     5 
1.1. Primary Aim  
 
 
 
 
 
 
 
 
 
     5 
1.2. Secondary Aims 
 
 
 
 
 
 
 
 
 
     5 
1.3. Hypothesis 
 
 
 
 
 
 
 
 
 
 
     5 
 
2. BACKGROUND  
 
 
 
 
 
 
 
 
 
     6 
2.1. Methotrexate Pharmacokinetics  
 
 
 
 
 
 
 
     6 
2.2. Polymorphisms  
 
 
 
 
 
 
 
 
 
     7 
2.3. Methotrexate Toxicity  
 
 
 
 
 
 
 
 
     7 
2.3.1. Nephrotoxicity  
 
 
 
 
 
 
 
 
     8 
2.3.2. Neurotoxicity  
 
 
 
 
 
 
 
 
     8 
2.3.3. Other side effects 
 
 
 
 
 
 
 
 
     8 
2.3.4. Management of Toxicity 
 
 
 
 
 
 
 
     8 
2.4. Rationale for Treatment Design  
 
 
 
 
 
 
 
     9 
 
3. STUDY ENROLLMENT AND ELIGIBILITY  
 
 
 
 
 
 
    11 
3.1. Study Enrollment    
 
 
 
 
 
 
 
 
    11 
3.2. Patient Eligibility Criteria  
 
 
 
 
 
 
 
 
    11 
 
4. TREATMENT PLAN  
 
 
 
 
 
 
 
 
 
    12 
4.1. Overview of Treatment Plan  
 
 
 
 
 
 
 
    12 
4.2. Study Endpoints   
 
 
 
 
 
 
 
 
 
    14 
 
5. EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED  
 
 
 
    15 
5.1. Physical Exam  
 
 
 
 
 
 
 
 
 
    15 
5.2. Laboratory Tests Prior to Treatment  
 
 
 
 
 
 
    15 
5.3. Demographic Information   
 
 
 
 
 
 
 
    15 
5.4. Laboratory Tests During Treatment  
 
 
 
 
 
 
    15 
5.5. Host Polymorphisms  
 
 
 
 
 
 
 
 
    15 
5.6. Other Information to be collected 
 
 
 
 
 
 
 
    16 
5.7. After Treatment Adverse Effects 
 
 
 
 
 
 
 
    16 
5.8. Shipping of samples  
 
 
 
 
 
 
 
 
    17 
 
6. DRUG INFORMATION   
 
 
 
 
 
 
 
 
    17 
 
7. CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA     18 
7.1. Removal from Protocol Therapy 
 
 
 
 
 
 
 
    18 
7.2. Off Study Criteria 
 
 
 
 
 
 
 
 
 
    18 
 
8. DATA MONITORING  
 
 
 
 
 
 
 
 
 
    18 
8.1. Stopping rules  
 
 
 
 
 
 
 
 
 
    18 
 
9. STATISICAL CONSIDERATION  
 
 
 
 
 
 
 
    18 
9.1. Statistical design  
 
 
 
 
 
 
 
 
 
    18 
9.2. Patient Accrual and Expected Duration of Trial  
 
 
 
 
 
    19 
9.3. Statistical Analysis Methods  
 
 
 
 
 
 
 
    19 
 
10. REFERENCES   
 
 
 
 
 
 
 
 
 
    20 


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APPENDIX A – Data to be Accessioned  
 
 
 
 
 
 
 
    23 
 
APPENDIX B – Suggested methotrexate infusion guidelines 
 
 
 
 
    24 
 


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1. 
GOALS AND OBJECTIVES 
1.1 
Primary Aim 
 
To determine the incidence of successfully achieving peripheral blood concentration of methotrexate of 
50-80µM in patients at high risk of drug toxicity after a 24 hour infusion of high-dose methotrexate 
treated according to our institutional treatment algorithm for individualized methotrexate dosing. 
 
1.2 
Secondary Aims 
1.2.1 To determine the incidence of the following side effects in high risk patients receiving high 
dose methotrexate as a 24 hour infusion: nephrotoxicity, neurotoxicity, mucositis, 
hepatotoxicity, and myelosuppression. 
1.2.2 To describe host polymorphisms that may identify risk of methotrexate toxicity or success of 
this institutional protocol. 
1.2.3 To describe predictors of successfully achieving an optimum concentration of methotrexate 
at the end of a 24 hour infusion of HDMTX in patients at high risk of drug toxicity treated 
with this individualized high dose methotrexate protocol.  
 
1.3 
Hypothesis 
At least eighty percent of patients with a high risk for or past history of significant methotrexate toxicity, 
receiving 24-hour high-dose methotrexate infusions per this  institutional algorithm for individualized 
dosing will have an appropriately therapeutic but minimally toxic 24 hour methotrexate level between 
50-80µM.   


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2. 
BACKGROUND 
Methotrexate is a crucial chemotherapeutic agent used to treat children with acute lymphoblastic 
leukemia (ALL), and is a staple of treatment regimens both nationally and internationally1.  In fact, 
cure rates for ALL approach 80%, in part due to the incorporation of methotrexate into treatment 
protocols1.  While all serum concentrations of methotrexate have shown to be efficacious in ALL 
therapy, when doses above 1g/m2 – considered high dose methotrexate (HDMTX) -  are given, 
several leukemic cell methotrexate resistance mechanisms are readily overcome2.  Most current 
protocols using HDMTX, including those run by the Children’s Oncology Group (COG) now utilize 
5g/m2 given as a 24 hour infusion in non-Down syndrome leukemia patients3.  Many studies have 
demonstrated the clinical benefits of HDMTX4;5, even when directly compared to lower doses of 
methotrexate6.  Recent data from the National Cancer Institute (NCI) sponsored COG trial AALL0232 
compared the effectiveness of HDMTX and 6-MP  against Capizzi escalating methotrexate plus PEG 
asparaginase (C-MTX/ASNase) in patients less than 30 years old with high-risk ALL (HR-ALL).  Initial 
analysis showed that patients randomized to receive HDMTX had significantly higher 5-year event-
free survival (EFS) compared to patients who received C-MTX/ASNase (82+ 3.4% versus 75.4 + 3.6%, 
p=0.006)7.  This finding lead to all patients with HR-ALL being treated with HDMTX.   
 
However, treatment with HDMTX can be associated with significant toxicity. The risk of morbidity and 
mortality from methotrexate toxicity increases when the drug is given at high dose.  Toxicities are 
directly correlated with increased drug exposure and since methotrexate is cleared renally, patients 
with kidney dysfunction are at particularly high risk of complications8.  In patients treated with HDMTX, 
1.8% will experience renal dysfunction9.  Patients with relapsed ALL are at increased risk of 
methotrexate toxicity due to prior exposure to nephrotoxic chemotherapy, as are patients with renal 
injury.  Per current COG protocols, patients with certain features indicating a high risk of toxicity to 
methotrexate will either have their methotrexate arbitrarily reduced by 25%, or it will be omitted from 
their treatment regimen entirely reducing or eliminating the important anti-leukemic effects of the drug. 
 
2.1  Methotrexate Pharmacokinetics 
 
The pharmacokinetics of methotrexate vary greatly among patients, with a fixed dose resulting in a 7-
fold variation in the range of plasma concentration5.  This variability can also exist in individual 
patients on subsequent administrations of HDMTX5.  Therapeutic drug monitoring has thus become a 
standard part of protocols in patients receiving HDMTX.   
 
Typically pharmacokinetics of HDMTX are described using a two compartment model with four 
parameters (CL – clearance, V1 – central compartment volume of distribution, V2 peripheral 
compartment volume of distribution, and Q – intercompartmental clearance). Clearance (CL) of MTX 
ranges from 3.5-7.1 L/h, and is inversely proportional to the age of the patient.  Among patients with 
the same total bodyweight (TBW), MTX clearance was 30% higher in children less than 10 years 
when compared with children greater than 10 years of age.  The volume of distribution of central 
compartment (V1) is 45% of TBW regardless of age10.   
 
The variation in pharmacokinetics also has important clinical implications.  Prospective 
pharmacodynamic studies of patients with ALL receiving HDMTX have shown that those patients with 
median MTX plasma concentration at steady state (Cpss) less than 16µM had significantly lower rates 
of remaining in remissions compared to patients with Cpss greater than 16µM5.   
 
 


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2.2  Polymorphisms 
 
High-dose methotrexate administered as a 24 hour infusion is a standard therapy for patients with 
leukemia, types of lymphoma, and other malignancies.  Though thousands of patients receive the 
same treatment, only a small subset will develop methotrexate toxicity.  This suggests that individual 
variations in methotrexate metabolism may contribute to the development of toxicities.   
 
Previous studies have identified several functional polymorphisms in both the folate cycle and 
methotrexate transporters now thought to play a significant role in the large variation in 
pharmacokinetics, response, and toxicity seen in patients who receive HDMTX11-13.  For example, the 
solute carrier 19A1 (SLC19A1, also known as reduced folate carrier 1, RLF1), is the main mechanism 
by which MTX enters the cell14.  The expression of SLC19A1 correlates with intracellular MTX 
concentration.  Variations in the SLC19A1 gene have been shown to impact event-free survival15.  
The most common single nucleotide polymorphism (SNP) in SLC19A1 results in the substitution of Arg 
with His at position 27.  Recent data have shown that this SNP is also associated with HDMTX 
toxicity12.   
 
Another example is found in individual variances in the enzyme methylenetetrahydrofolate reductase 
(MTHFR).  Once MTX is transported into the cell, it is metabolized to polyglutamylated metabolites.  
These metabolites inhibit enzymes involved in the folic acid cycle, including MTHFR.  MTHFR plays 
an important role in the folic acid cycle as it is responsible for catalyzing the reduction of 5, 10-
methylenetetrahydrofolate to 5-methyltetrahydrofolate16.  A common cysteine to threonine substitution 
at MTHFR 677 results in lower enzyme activity and has been shown to be associated with decreased 
HDMTX clearance and increased toxicity12.  SNPs associated with SLCO1B1, an organic anion 
transporter polypeptide, have also been shown be involved in MTX clearance13.   
 
2.3 
Methotrexate Toxicity 
While integral to improving the survival of patients with malignancy, HDMTX can also result in severe 
treatment-related complications.  The main side-effects of HDMTX treatment include mucositis, 
hepatitis, renal dysfunction, prolonged myelosuppression, CNS toxicities such as seizures and stroke-
like syndromes, and dermatitis17;18.  In order to minimize these side effects, drug levels are closely 
monitored, and aggressive supportive care measures are standard practice.  During HDMTX 
infusions, hyper-hydration and alkalization of urine promote efficient clearance of the drug. The 
tetrahydrofolic acid derivative leucovorin is given post-infusion to rescue normal DNA synthesis in non-
leukemia cells minimizing myelosuppression19.  Leucovorin dosing is typically based on post infusion 
methotrexate concentrations and increased for slow methotrexate clearance.    
 


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2.3.1 Nephrotoxicity 
As methotrexate is excreted nearly exclusively by the kidneys, methotrexate induced nephrotoxicity is 
a serious potential complication of treatment20.  The two main mechanisms of methotrexate induced 
nephrotoxicity involve damage to the renal tubules via either a direct toxic effect21 or as a result of the 
precipitation of methotrexate metabolites22;23.  The two main methotrexate metabolites, 7-hydroxy-
methotrexate (7-OH-MTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA), are at least six times 
less soluble at acidic pH than methotrexate22;24.  This decreased solubility prompted alkalinization of 
the urine to be a major component of methotrexate supportive care.  In addition, alkalinization forces 
the ionization of methotrexate, a weak acid, making it more water soluble, preventing precipitation and 
trapping unchanged cleared methotrexate in urine for efficient elimination. 
In patients receiving HDMTX, 1.8% will experience renal dysfunction resultant from HDMTX.  This 
high risk group of patients has a mortality rate of 4.4%, greater than the mortality rate of 0.8% among 
patients who did not develop HDMTX induced renal dysfunction9.   
 
2.3.2 Neurotoxicity 
 
Methotrexate toxicity can lead to severe neurological sequelae.  Among children treated with 
methotrexate, 3-11% will experience either transient or chronic symptoms of neurotoxicity such as: 
seizures, hemiparesis, aphasia, headache, and altered mental status25.  However, up to 76% of 
patients treated with HDMTX demonstrate leukoencephalopathic changes on magnetic resonance 
imaging (MRI)26. The prevalence of leukoencephalopathy increases with each subsequent course of 
HDMTX.  Among patients who developed leukoencephalopathy following HDMTX, 10-56% had their 
symptoms recur when re-challenged with HDMTX27-29.     
 
2.3.3 Other side effects 
 
Other common side effects of HDMTX include mucositis, hepatitis, and prolonged myelosuppression.  
These side effects can result in hospital admission for management of symptoms, as well as a delay in 
subsequent cycles of chemotherapy.  Increased time between chemotherapy cycles may contribute 
to inferior outcomes30;31.  Delayed clearance of MTX is thought to contribute to the development of 
these side effects, as the time of exposure to MTX is prolonged19.   
 
2.3.4   Management of Toxicity 
 
As methotrexate is primarily cleared via renal excretion, patients with underlying renal dysfunction are 
at an increased risk of having delayed clearance.  The elevated and prolonged exposure to 
methotrexate that results from delayed clearance makes patients more susceptible to developing other 
drug related complications such as mucositis and myelosuppression32.   
 
In an effort to minimize the nephrotoxic effect of HDMTX, patients concurrently receive leucovorin, 
hydration, and alkalinization19.  If nephrotoxicity and delayed clearance develop despite these 
measures, two further options exist for lowering plasma methotrexate concentrations: dialysis and 
glucarbidase (CPDG2).  After dialysis plasma MTX concentrations decrease by a median of 52% 
(range 26-82%).  Cases of rebound increases in MTX plasma concentrations by 10-221% have been 
reported.  Patients are also subjected to the risks of dialysis including bleeding, infection, and 
electrolyte disturbances8.  The total cost through hospital discharge for patients receiving dialysis can 
range from $94,000 to $140,00033.   


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The enzyme CPDG2, which hydrolyzes the terminal glutamate from naturally occurring folates and 
folate analogs such as methotrexate, is a well-tolerated alternative to lowering plasma MTX levels3.  
Within 15 minutes of administration, MTX plasma concentrations in patients with osteosarcoma, non-
Hodgkin lymphoma, or ALL decreased by a median of 98.7% (range 95.6-99.6%).  A rebound 
increase of 2.8-8.8% of the MTX concentration was seen in 60% of patients9.  The drug cost of 
CPDG2 for a 50kg patient is approximately $30,000, excluding costs associated with preparation, 
administration, and pharmacy overhead (personal communication, Brooke Bernhardt, PharmD, BCOP, 
COG pharmacist and clinical pharmacy specialist, Texas Children’s Cancer Center).  In addition, 
despite the fact that is recommended to consider CPDG2 in patients with sustained MTX 
concentrations of > 10 M at 42-48 hours after the start of a MTX infusion, it is currently a highly 
regulated, difficult to obtain medication8.  Although it is now commercially available, it requires special 
order from the manufacturer (http://www.btgplc.com/products/specialty-pharmaceuticals/voraxaze).  
In addition, minimal dosing is kept available by most tertiary care hospitals.  Texas Children’s 
Hospital, for example, keeps four 1000 unit vials on site. This is enough for one time dosing for an 
80kg patient (personal communication, Amanda Berger, PharmD, Texas Children’s Cancer Center).   
  
There is currently no standardized method of administering HDMTX to patients at risk for increased 
methotrexate toxicity.  As a result, these patients are at risk of having HDMTX removed from their 
treatment regimen or suffering severe morbidity from its side-effects.  Current COG protocols suggest 
postponing and/or eventually omitting HDMTX administration for impaired renal function, grade 3-4 
mucositis, hepatitis, and/or prolonged myelosuppression.  In patients who experience and fully 
recover from these effects, the current COG recommendations are to reduce subsequent doses of 
HDMTX.  As methotrexate metabolism varies widely among individuals, a fixed dose-reduction 
without respect for individual patient characteristics results in patients being at risk for either not 
reaching a therapeutic level of methotrexate or experiencing further drug-related side effects.   
 
2.4  Rationale for Treatment Design 
 
The incorporation of HDMTX into treatment regimens for pediatric patients with ALL has improved cure 
rates now approaching 80%.  HDMTX has proven to be effective in treating ALL, regardless of 
prognostic factors such as age, immunophenotype, and genetics1.  HDMTX is also effective at 
treating other malignancies such as Burkitt’s lymphoma and mixed-phenotype leukemia, among others.  
Recent data from the NCI-sponsored COG clinical trial AALL0232 has shown that patients with HR-
ALL have significantly improved EFS when treated with HDMTX/6-MP as compared to C-
MTX/ASNase7.  As a result of this finding, patients with HR-ALL are no longer randomized to 
treatment during Interim Maintenance.  Instead, all patients who are able receive HDMTX during 
interim maintenance. 
 
Every effort should thus be made to ensure that patients can safely receive HDMTX.  As effective as 
it is in treating leukemia, administration of HDMTX can result in serious side effects such as renal 
dysfunction, neurotoxicity, mucositis, prolonged myelosuppression, hepatitis, and dermatitis. The risk 
of methotrexate toxicity is increased in patients with impaired renal function.  Due to underlying 
medical conditions and prior cytotoxic chemotherapy, a subset of patients with ALL is at an increased 
risk of developing severe methotrexate-related toxicity.  There is currently no standardized method of 
administering HDMTX to patients at risk in increased methotrexate toxicity.  As a result, these 
patients are at risk of having HDMTX removed from their treatment regimen or suffering severe 
morbidity from its side-effects.   
 
Prior studies, mostly at the St. Jude Children’s Research Hospital (SJCRH) have shown that 


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individualized methotrexate protocols can be safe and effective34;35.  Wall and colleagues used real-
time complex pharmacokinetic modeling to dose-adjust HDMTX in the treatment of patients with 
relapsed ALL.36.  The group demonstrated that patients receiving HDMTX have an average clearance 
of 103ml/min/m2 34, which corresponded to a steady-state plasma concentration of (Cpss) of 65µM + 
10µM at 24 hours35.  In addition, 58% of their patients required dose-adjustments to achieve a Cpss 
within this range.  None of their patients experienced severe methotrexate-related toxicity.  They 
found that the initial methotrexate clearance was the best predictor of high methotrexate Cpss36.   
 
Based largely on the previous work at SJCRH, we developed an individualized methotrexate treatment  
plan as an option to safely administer HDMTX, with a target Cpss of 65 M to patients at risk for 
delayed clearance and subsequent toxicity.  Other options for this population would include a 25% 
methotrexate dose reduction or complete omission of HDMTX from their treatment.  Since 2007, 
Texas Children’s Hospital (TCH) has given patients with past history of toxicities including renal 
toxicities individualized methotrexate with no known increase in toxicities based on evolving versions 
of this treatment plan (n =14 patients; ref: personal communications with faculty members of the Texas 
Children’s Hospital Leukemia Program).  Work by Aumente, et al, demonstrated that the Monte Carlo 
simulated MTX concentration and means over time show a peak methotrexate concentration near 
hour 2, with steady state occurring near hour 610. Therefore, our treatment plan provides guidelines for 
HDMTX dose adjustments based on hour 2 and 6 methotrexate levels.  In addition, these 
adjustments are based on a fixed, simple bed-side algorithm and thus avoid the need for complex 
pharmacokinetic modeling.    Patients treated according to the algorithm may thus not need their 
dose altered.  With this in mind, in addition to our eligibility criteria we will allow attendings to enroll 
patients as they deem necessary, since if the MTX Cpss is therapeutic no alterations to therapy will be 
made.  In September, 2012, this treatment plan was formalized and approved for clinical use by the 
Texas Children’s Cancer Center Chemotherapy Safety Committee.   
 
In addition, despite the fact that MTX pharmacokinetics, tumor response and patient toxicities are 
known to vary greatly and it is known that several germline polymorphisms play a role in this variation, 
much controversy about the functional consequences of these polymorphisms still exists11.  Our study, 
will, by definition, isolate a population of patients with a known increased propensity for certain MTX 
toxicities.  Because the general population minor allele frequency (MAF) is known for many of the 
polymorphisms known to affect MTX metabolism, this study will provide a unique opportunity to 
compare rates of certain of these polymorphisms in our study population to that of the general 
population.  This may help elucidate which polymorphisms truly correlate to deficiencies in MTX 
metabolism.  In addition, we may be able to discover which polymorphisms describe a population 
best suited for individualized MTX dosing.      
 
Though many genetic targets in the folate cycle and intracellular MTX transportation have already 
been identified, new target genes continue to be discovered.  Given that the knowledge base in this 
field is rapidly expanding, the decision of which polymorphisms to be tested will be determined at the 
time of analysis, when the study accrual has been met.  Common SNPs currently thought to be 
involved in methotrexate metabolism, pharmacodynamics and toxicity are listed below: 
 
Polymorphism 
SNP effect 
Approximate 
MAF (%)
Clinical effect 
Reference 
MTHFR 677C>T 
Reduced activity 
15-25
Both increased and decreased 
risk of myelosuppression 
reported 
37;38 
SLC19A1 80G>A 
Reduced activity 
30-50
Increased hepatotoxicity 
39 
MTHFR 1298A>C 
Reduced activity 
15-20
Decreased myelosuppression 
40 
TYMS 28-bp triple 
repeat 
Increased 
transcription 
40-50
None reported 
41 


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This research does not qualify for an IND as the use of HDMTX is per standard of care treatment of 
ALL, and the route of administration or dosage level does not significantly increase risk to the patient. 
3. 
STUDY ENROLLMENT AND PATIENT ELIGIBILITY 
3.1 
Study Enrollment 
3.1.1 IRB approval 
Approval for the use of this protocol by the individual institution's Human Subjects Review 
Committee must be obtained, in accordance with the institutional assurance policies of the U.S. 
Department of Health and Human Services.  
3.1.2 Study Enrollment 
Patients may be enrolled on study once all eligibility requirements for the study have been met.  
Patients will be enrolled by calling Kathy McCarthy at 832-824-4804 Mon-Fri from 9am-5pm 
central time.  At enrollment patients will be assigned an individual identification number.   
 
3.1.3 Timing 
Study enrollment must take place prior to patients receiving methotrexate dose modifications. 
 
3.2 
Patient Eligibility Criteria 
 
INCLUSION CRITERIA 
3.2.1 Age 
Patients must be ≥365 days and <23 years of age at the time of enrollment. 
3.2.2 Diagnosis 
Patients  with any malignancy who will receive HDMTX given as a 5 g/m2 infusion over 24 hours 
and a history of ≥1 of the following: 
 
Documented decreased renal function, as defined as Creatinine greater than 1.5 x 
baseline or GFR <65ml/min/1.73m2. 
 
History of prior nephrotoxicity with HDMTX as evidence by increased creatinine to 
1.5 x baseline or need for dialysis or carboxypeptidase 
 
History of Grade 3 adverse event (AE) related to HDMTX (mucositis, 
myelosuppression, nephrotoxicity, hepatotoxicity) based on the NIH Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 
 
Provider concern patient is at risk for MTX toxicity, such as a prior history of 
treatment with nephrotoxic chemotherapy, history of HDMTX-related neurotoxicity, or 
antimicrobial/antifungal therapy 
 
 


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EXCLUSION CRITERIA 
3.2.3 Unable to draw labs for HDMTX serum concentration 
3.2.4 Enrollment on a protocol (COG or other) which restricts proposed dose modifications 
 
3.2.5 Patients with Trisomy 21 
 
3.2.6 Patients with > grade 1 neurologic toxicity at the time of enrollment that is attributed to 
unresolved prior methotrexate toxicity 
 
3.2.7 Patients with ≥ grade 3 chronic kidney disease at enrollment (eGFR or CrCl < 
30ml/min/1.73m2) 
 
REGULATORY 
3.2.8 Informed consent for participation in this study must be obtained within 30 days from the start 
of HDMTX on this protocol and prior to any research procedures from the patient and/or the patient's 
legally authorized guardian in accordance with institutional policies approved by the U.S. Department 
of Health and Human Services 
3.2.9 All institutional, FDA, and NCI requirements for human studies must be met.  
 
4. 
TREATMENT PLAN 
 
4.1 
Overview of Treatment Plan 
Our prospective study will enroll patients who will receive HDMTX given as a 5g/m2 24-hour infusion.  
Enrolled patients will be receiving HDMTX as determined by their primary provider.  Patients are eligible 
to be enrolled at any time point during their treatment course, and may participate during any one or more 
of their multiple HDMTX administrations.   
 
Concomitant Medication Restrictions: 
1.  Sulfamethoxazole and Trimethoprim (SMX-TPM); synonyms: Bactrim, Co-trimoxazole 
2.  Non-steroidal anti-inflammatory medications (NSAIDS) 
3.  Penicillins 
4.  Proton pump inhibitors 
5.  Aspirin-containing medications 
 
Hold the above medications on the day of HDMTX infusion and for at least 72 hours after the start of 
the HDMTX infusion and until the MTX level is less than 0.4µM (In the presence of delayed clearance 
continue to hold these medications until MTX level is less than 0.1 µM):  
 
As per the Children’s Oncology Group (COG) and institutional guidelines, restricted medications 
should be stopped (i.e. 1.  Sulfamethoxazole and Trimethoprim (SMX-TPM); synonyms: Bactrim, Co-
trimoxazole; 2.  Non-steroidal anti-inflammatory medications (NSAIDS); 3.  Penicillins; 4.  Proton 
pump inhibitors; 5.  Aspirin-containing medications), and prior to receiving HDMTX patients will 
receive IV hydration with D5 ¼ NS with NaHCO3  3 mEq/100 mL at 200 mL/m2/hr for a minimum of 2 
hours, until urine pH ≥ 7.0 and ≤ 8.0 and urine specific gravity ≤1.010. 
 


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13 
Once these parameters are met, between hours 0-1 of the protocol patients will receive methotrexate 500 
mg/m2 in D5 ¼ NS with NaHCO3 3 mEq/100 mL at 200 mL/ m2 IV over 60 minutes.  This will be followed 
immediately by Hours 1 to 24 Methotrexate 4500 mg/m2 in D5 ¼ NS with NaHCO3 3 mEq/100 mL 4600 mL/ m2 
IV at 200 mL/m2/hr for 23 hours. 
 
Dose adjustments will be made based on the following tables.  Adjustments should be made as soon as 
possible after MTX levels checked are resulted.  Interventions are expected to be done within 120 minutes of 
sending the MTX level. 
A. Interventions at 2 hours  
 
If MTX level 
is: 
<100µM 
≥100µM 
MTX 
administration 
intervention 
·Continue unchanged 
·Stop infusion for 1 hour  
·At restart time; restart MTX at 50% reduction 
from original infusion rate; therefore, restart 
at 100 mL/m2/hr 
Fluid 
intervention 
·Continue unchanged 
·At restart time; begin D5 ¼ NS with 
NaHCO3 3 mEq/100 mL at 100 mL/m2/hr) 
MTX level 
specific lab 
instructions 
·Check 6 hour MTX level and again at 
24, 36, 42 and 48 hours per COG 
algorithm# 
·Check 8 hour MTX level and then per the 
COG algorithm# at 24, 36, 42 and 48 hours* 
 
B. Interventions at 6 hours (*8 hours if 2 hour level was ≥100µM) 
 
If MTX level 
is: 
<75µM 
≥75µM/<100µM 
≥100µM 
MTX 
administration 
intervention 
·Continue  
Unchanged 
·Reduce MTX infusion 20% 
from MOST RECENT infusion 
rate; Therefore, if (select A OR 
B): 
A) 2 hr MTX level was <100 
M and therefore, infusion rate 
was 200 mL/m2/hr then restart 
at 160 mL/m2/hr 
B) 2 hr MTX level was ≥100 
M and therefore, infusion rate 
was 100 mL/m2/hr then restart 
at 80 mL/m2/hr 
·Stop infusion for 1 hour  
·At restart time; restart MTX at 
50% reduction from MOST  
RECENT infusion rate; 
Therefore, if (select A OR B):  
A) 2 hr MTX level was <100 µM 
and therefore, infusion rate was 
200 mL/m2/hr then restart at 
100 mL/m2/hr 
B) 2 hr MTX level was ≥100 µM 
and therefore, infusion rate was 
100 mL/m2/hr then restart at 50 
mL/m2/hr 
Fluid 
intervention 
·Continue 
unchanged 
Select A OR B: 
A) If 2 hr MTX level was <100 
µM and therefore, there was 
no previous additional fluid 
order then begin D5 ¼ NS with 
NaHCO3 3 mEq/100 mL at 40 
mL/m2/hr 
B) If 2 hr MTX level was ≥100 
µM and therefore, additional 
D5 ¼ NS with NaHCO3 3 
mEq/100 mL was already 
running at 63.5 mL/m2/hr then 
increase this fluid to run at 120 
mL/m2/hr 
Select A OR B 
A) If 2 hr MTX level was <100 
µM and therefore, there was no 
previous additional fluid order 
then begin D5 ¼ NS with 
NaHCO3 3 mEq/100 mL at 100 
mL/m2/hr 
B) If 2 hr MTX level was ≥100 
µM and therefore, additional D5 
¼ NS with NaHCO3 3 mEq/100 
mL was already running at 100 
mL/m2/hr then increase this 
fluid to run at 150 mL/m2/hr 


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14 
MTX level 
specific lab 
instructions 
·Check MTX level 
again at 24, 36, 42 
and 48 hours per 
COG algorithm# 
·Check MTX level again at 24, 
36, 42 and 48 hours per COG 
algorithm# 
·Check MTX level again at 24, 
36, 42 and 48 hours per COG 
algorithm# 
#see COG algorithm on page 21 
 
Subjects will come off protocol therapy for infusion delays longer than 2 hours.  In addition, the total 
methotrexate infusion time, on this study, should be no longer than 26 hours.  Infusions should only 
be extended past 24 hours to accommodate stops for other med infusions.  Increases in rate of MTX 
infusion to ensure completion within this time frame are not allowed. 
 
The interventions in this protocol occur at the 2 and 6 hour (or 8 hour if the 2 hour level was ≥100µM) 
time points.  After the 24hr HDMTX infusion is complete, the individualized protocol will end and 
patients will remain on study but be off of the protocol therapy.  After this time, management of MTX 
levels will continue according the treating physician’s discretion using standard supportive care 
guidelines.  This is elaborated below in Appendix B, suggested supportive care guidelines based on 
the COG algorithm. Interventions for toxicity are at the discretion of the treating provider as patients 
will be off treatment protocol at the 24hr time point. Glucarpidase should be administered based on the 
treating physician’s discretion, with the recommendation that it be administered prior to the 
development of grade 4 nephrotoxicity and prior to 96 hours after the start of the HDMTX infusion.  
Administration of CPDG2 after these time points have been shown to be statistically associated with 
the development of ≥ grade 4 toxicity3. 
 
 
Patients may be re-enrolled on this protocol therapy again for subsequent HDMTX infusions without 
the need for re-consent. In order to re-enroll, call the study coordinating center as outlined in Section 
3.1 PRIOR to the cycle in which the dose modification protocol will be implemented. 
 
The active length of study participation for each patient is 4 weeks after the last high-dose 
methotrexate infusion or until the next cycle of chemotherapy begins (which ever occurs first).  
Therefore, the maximum amount of active time on study would be 2.5 months.  This would include 4 
doses of HDMTX given every other week plus a 4 week follow-up period to assess for side effects. 
 
 
4.2 
Study Endpoints 
After the initial monitoring of MTX levels and creatinine during the MTX infusion, patients will be 
followed for the development of HDMTX related toxicity for 4 weeks after their infusion or until their 
next cycle of chemotherapy begins, whichever is shorter.   
 


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15 
5. 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
At Study Entry 
5.1 
Physical Exam 
5.2 
Laboratory Tests Prior to Treatment 
 
A maximum of 28 days prior to the first dose of HDMTX on this protocol a nuclear 
medicine GFR must be performed.  
 
A maximum of 7 days prior to the first dose of HDMTX on this protocol the following labs 
will be collected (Week 0 labs): CBC, Chem 10, AST, ALT, total and direct bilirubin, and 
10 mL in a purple top (minimum of 5mL will be accepted) of additional blood for MTX 
transportation genetic analysis.  Sample for MTX transportation genetic analysis is 
optional. 
 
5.3    Demographic information 
 
Age, race, leukemia type, cytogenetics of leukemia, leukemia risk stratification, disease status, 
protocol treated according to, concomitant medication, reason for enrollment on protocol and type of 
adverse event with prior HDMTX. 
5.4 
Laboratory Tests During Treatment 
 
Methotrexate levels and creatinine at times 2, 6 (or 8) and 24 hours are required for 
research purposes.  Additional methotrexate levels and creatinine should be performed 
per institutional standards.  Methotrexate levels and creatinine at hours 36 (if 
methotrexate levels are ≥150 micromolar), 42, 48 and every 6-12 hours thereafter until 
methotrexate is considered cleared (e.g. ≤0.4 micromolar at hour 48 and/or <0.1 
micromolar thereafter) is highly recommended but considered optional.  An example of 
routine post-methotrexate monitoring is outlined in Appendix B.  
 
The following labs should be drawn at Week 1 and Week 2 and repeated weekly until 
either the next dose of HDMTX is administered or 4 weeks have passed since the last 
dose of HDMTX: CBC, Creatinine, BUN, AST, ALT, total and direct bilirubin. 
 
5.5 Host Polymorphisms 
 
The literature on SNPs influencing the pharmacokinetics and pharmacodynamics of methotrexate is 
rapidly evolving11.  Based on current knowledge, at minimum we will assay each participant who 
consents for the SLC19A1 80G>A, TYMS 28-bp triple repeat, MTHFR 677C>T and MTHFR 1298A>C 
SNPs.  In addition, technologies for genotype analysis are constantly changing and improving.  
However, for the above SNPs multiple technologies have been discovered and validated12. Our 
samples will be batched, and the most appropriate technique for genotyping at the time of analysis will 
be performed.  Our collaborator, Dr. Donald W. (Will) Parsons has vast expertise in various 
genotyping methods including TaqMan, restriction enzyme, and Sanger sequencing.   
 
5.5.1 Genotyping Methods 
More than one genotyping technique may need to be used for different samples.  Dr. Parsons’ lab 
has experience in this field, and will be able to perform the necessary technique to achieve 
reproducibility in all samples.  When choosing a method we will consider, cost, amount of DNA 


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needed, and availability of technique. 
 
5.5.2 DNA Extraction and Distribution 
Five to 10 mL of peripheral blood will be collected for genotyping purposes prior to the initiation of 
the methotrexate infusion, during the gathering of additional baseline laboratories.  At this time 
additional baseline labs are routinely collected and peripheral blood mononuclear cells will be 
optimally present prior to methotrexate/chemotherapy induced myelosuppression.  Mononuclear 
cells will be separated from whole blood using Ficoll-Hypaque centrifugation.  DNA will be isolated 
from the enriched mononuclear cells using the QIAgen DNeasy kit per the manufacturer’s 
instructions.  To avoid DNA loss or degradation DNA samples will be stored in Axygen or 
comparable tubes.  Based on prior experience with ALL samples, we expect an average of 
18.5mcg of DNA per sample.  Most current genotyping techniques require 5-20ng per assay. 
 
 
5.6    Other information to be collected 
 
 
Ordered HDMTX dose 
 
date/time of 24hr infusion start 
 
initial HDMT infusion rate with HDMTX concentration 
 
date/time of each change in HDMTX infusion rate and rate at which new infusion is set (if 
applicable) 
 
date/time of 24hr infusion start and stop for concomitant medications 
 
date/time of completion of HDMTX infusion 
 
toxicities experienced during infusion,  
 
hour at completion of monitoring 
 
total MTX given 
 
total leucovorin given 
 
all MTX levels resulted and time each MTX level was drawn. 
 
 
 
5.7 
After Treatment Adverse effects 
 
Toxicities will be assessed until the next cycle of chemotherapy is administered or until 4 weeks after 
the administration of HDMTX, whichever time period is shorter.  Each grade >3 adverse event will be 
collected for the following methotrexate related toxicities: mucositis, myelosuppression, hepatotoxicity 
(AST, ALT, GGT, bilirubin).  All adverse events will be collected for neurotoxicity and nephrotoxicity.  
If multiple occurrences of a toxicity take place during a single course, please list a separate Max 
Grade This Occurrence for each occurrence. 
 
The International Conference on Harmonization (ICH) Guideline for Good Clinical Practice E6(R1) 
defines an adverse event (AE) as: 
 
“Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this treatment.  
An AE can therefore, be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal product, whether or 
not considered related to the medicinal product.” 
 
A Serious Adverse Event (SAE) is any AE that: 1.  Results in death; 2.  Is immediately life 
threatening; 3.  Results in hospitalization – either initial or prolonged; 4. Results in persistent or 
significant disability/incapacity; 5.  Results in congenital anomaly or birth defect; 6.  Results in 
required intervention to prevent permanent impairment or damage or 7.  Results in another serious 


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important medical event. 
 
For the purposes of this study, it is not required to report SAEs which result in admissions to the 
hospital for simple febrile neutropenia or prolongation of a hospitalization secondary to delayed 
methotrexate clearance. 
 
All SAEs should be identified and reported locally per institutional standards.  
 
All unanticipated problems involving risks to subjects or others (UPIRSOs) should be identified and 
reported locally per institutional standards. 
 
All reportable AEs and SAEs recorded during this study will be summarized.  The incidence of 
treatment-emergent AEs (new or worsening from baseline) will be summarized by primary system 
organ class, severity based on CTCAE, version 4.0, type of adverse event and relationship to 
methotrexate.  Deaths reportable as a Serious Adverse Event (SAE) and non-fatal SAEs will be listed 
by patient and tabulated by primary system organ class and type of adverse event.  Any other 
information collected (e.g. start/end dates and duration of AE, severity or relatedness to methotrexate) 
will be listed as appropriate. 
 
All reportable SAEs and UPIRSOs should be reported to the Principal Investigator Jennifer Foster at 
jhfoster@txch.org and the Research Coordinator Kathy McCarthy at ksmccart@texaschildrens.org 
within 5 business days. 
 
5.8 
Shipping of samples 
Submit materials to: 
 
 
 
Linna Zhang 
 
 
 
1102 Bates St, Suite 1220.07 
 
 
 
Houston, TX 77030 
 
 
 
Ph 832-824-4592 
 
6. 
DRUG INFORMATION 
See the consent document for toxicities. All other information is available on the COG website in the 
manual titled ―Drug Information for Commercial Agents used by the Children‘s Oncology Group 
https://members.childrensoncologygroup.org/prot/reference_materials.asp under Standard Sections 
for Protocols. Also see drug package inserts for added information on toxicities. 


Protocol Version 8.0  
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18 
7. 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA  
7.1 
Removal from Protocol Therapy 
7.1.1 > Grade 3 renal or neurologic toxicity 
7.1.2 Parent/patient or provider request  
7.1.3 
Inability to analyze methotrexate levels at given time points.  Patients will come off of 
study secondary to the inability to make dose modification decisions if methotrexate 
levels at required time points are not available within 120 minutes of drawing the level.  
If this occurs, treatment decisions will be up to the treating physician.  Options would 
include: discontinuing the methotrexate infusion, altogether or calculating a 25% total 
dose reduction (as suggested by COG) and stopping the infusion when the total dose 
given reaches the newly calculated reduced dose. 
7.1.4 Methotrexate infusion interrupted for longer than 2 hours. 
 
7.2 
Off Study Criteria 
7.2.1 Lost to follow-up 
7.2.2 4 weeks after the end of methotrexate infusion or start of next chemotherapy, which ever is 
shorter. 
 
7.2.3 Death 
 
8. Data Monitoring 
 
Texas Children’s Cancer Center, where we will be conducting our research, is part of the Dan L. 
Duncan Cancer Center (DLDCC).  The NCI-designated Dan L. Duncan Cancer Center at Baylor 
College of Medicine is a consortium made up of several institutions across the Texas Medical 
Center.  As part of the DLDCC, we will have access to various shared resources, including a 
Clinical Trials Support Unit.  For our data monitoring, we will utilize the Data Safety and Monitoring 
Committee (DSMC) through the DLDCC. 
 
8.1. Stopping rules 
8.1.1. Two patients with HDMTX induced nephropathy necessitating dialysis or administration 
of carboxypeptidase. 
8.1.2. Two patients achieving a 24 hour Cpss of <20µM. 
8.1.3. Two patients needing intensive care admission for HDMTX related toxicities. 
8.1.4. Any death attributed to HDMTX toxicity. 
 
If any of the above criteria are met then the study will be stopped, the incident(s) will be reviewed, and 
the study will be amended to address the incident(s) prior to potentially re-opening. 
9. 
Statistical Considerations 
9.1 
Statistical Design 
Aim 1 1a) Calculate the incidence of success with the new protocol (achieving an end infusion 


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Date: June 17, 2015 
 
19 
peripheral blood MTX concentration between 50-80 μM) 
 
1b) Explore potential changes in incidence of success (achieving an end infusion peripheral 
blood MTX concentration between 50-80 μM) between sequential administrations of HDMTX.  The 
primary endpoint and sample size calculation refer to the first cycle for each patient. 
 
Aim 2 2a) Calculate the frequency of adverse effects after each infusion.  Success rate over multiple 
cycles within individual patients will be analyzed using generalized estimating equations for binary 
outcomes. This method will also be applied to the frequency of adverse events and to the assessment 
of factors associated with outcome. Continuous and ordinal/non-normal adverse effects will be 
compared via paired t-test or Wilcox sign rank test respectively. 
 
2b) Describe the genotype of various methotrexate metabolizing SNPs in each subject.  The 
polymorphisms of subjects in this study will be compared to published minor allele frequencies and 
when appropriate will be stratified by race/ethnicity.  The purpose of this analysis will be to describe 
trends away from the major allele frequency in patients known to either have experienced or who are 
at high risk for developing adverse effects to HDMTX.   
 
2c) Examine predictors of success of achieving a CPss of 50-80 μM.  Variables in our analysis 
will include (but not be limited to) age, sex, disease type, race/ethnicity and measure of pre-MTX renal 
function.  An analysis both with and without SNP genotype will be performed secondary to the 
likelihood that race/SNP correlations will interfere with SNPs being a truly independent variable.  We 
will initially use chi-square test for group variables and t-test for continuous variables.  Logistic 
regression will be used for multivariable analysis to control for confounders and estimation of adjusted 
odds ratios.  P<0.05 will be considered statistically significant.       
 
 
9.2 
Patient Accrual and Expected Duration of Trial 
Patients will be continually accrued until a target of 50 methotrexate infusions requiring any protocol-
prescribed dose adjustments are reached.  The expected duration of accrual for the trial is 2-2.5 
years. 
9.3 
Statistical Analysis Methods 
Sample size with power justification:  
 
Sample size is estimated on primary study objective of estimating the true rate of success with the 
new protocol. Success is defined as 80% of infusions achieving 24hr HDMTX end infusion MTX 
values of 50-80µM.  A sample size of 50 methotrexate infusions per the algorithm produces a two-
sided 95% confidence interval with a width equal to 0.22 when the sample proportion is 80%. We will 
be 95% confident that the true success rate in the study would be between 69% and 91%. 
 
Analysis:  
Aim 1a) Overall change in the incidence of success with the new protocol (achieving end infusion MTX 
levels 50-80µM) and 
1b) change in incidence of success at various infusion times during interim maintenance and 
interim maintenance-like cycles in which patients are administered HDMTX every 2 weeks, as well as  
1c) Success rate over multiple cycles within individual patients will be analyzed using generalized 
estimating equations for binary outcomes. This method will also be applied to the frequency of 
adverse events and to the assessment of factors associated with outcome. Continuous and 
ordinal/non-normal adverse effects will be compared via paired t-test or wilcox sign rank test 
respectively.   
 
Aim 2) To examine predictors of success (achieving CPss of 50-80µM) we will initially use chi-square 


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Date: June 17, 2015 
 
20 
test for grouped variable and t-test for continuous variables. Logistic regression will be used for 
multivariable analysis to control for confounders and estimation of adjusted odds ratios. P<.05 will be 
considered statistically significant. 
 
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APPENDIX A 
 
 
Schedule of Assessments 
 
 
Hours after methotrexate infusion 
starts (Week 0) 
Weeks 
 
Procedures 
S 
0 
2 
6 1 24 
36‡ 
42‡ 
482‡ 
1 
23 
Protocol 
section  
Blood for SNP analysis 
 
X 
 
 
 
 
 
 
 
 
5.5.2 
Nuclear eGFR  
X 
 
 
 
 
 
 
 
 
 
5.2 
CBC with diff and 
platelets 
 
X 
 
 
 
 
 
 
X 
X 
5.2  
BUN 
 
X 
 
 
 
 
 
 
X 
X 
5.2 
AST/ALT/T&D* bilirubin 
 
X 
 
 
 
 
 
 
X 
X 
5.2, 5.4 
Creatinine 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
5.4 
Methotrexate level 
 
 
X 
X 
X 
X 
X 
X 
 
 
4.1 
S: Screening; Week 0: Week of high dose methotrexate administration; 1Hour 6 or 8 depending on 2 hour 
methotrexate level (Sec 4.1); ‡ highly recommended but optional; should be done per institutional standards of 
post-methotrexate monitoring; 2 Continue 48 hour labs per algorithm (recommended but optional) (See appendix 
B); 3 Repeat Week 2 labs weekly until the next dose of methotrexate or 30 days since last dose of methotrexate; 
*T&D: Total and Direct 
 


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24 
Draw 24-hr MTX level 
 
MTX <150 µM 
MTX >150 µM 
or 
Cr > 125% baseline
Draw 36-hr MTX level++ 
MTX >3 µM 
MTX >10 µM 
Consider glucarpidase 
Increase hydration 
to 200 mL/m2/hr 
Draw MTX level (& Creatinine) 
at Hr 42++ & Hr 48 
Continue standard hydration 
(i.e @ 125 mL/m2/hr) 
MTX <3 µM 
Hr 42 ≤1.0 µM 
  
LCV 15 mg/m2 
at hrs 42, 48, & 
54, then stop 
LCV 
 
LCV 15 mg/m2 at 
hrs 42, 48, & 54 
and continue 
q6hrs until MTX 
level < 0.1 µM 
Hr 42 = 10-19.9 µM 
Hr 42 = 20-200 µM 
Hr 42 >200 µM 
 
Hr 42 = 1.01-9.9 µM 
 
LCV 15 mg/m2 
starting at hr 42 
and continue 
q3hrs, until MTX 
level < 0.1 µM 
 
Repeat MTX 
LCV 100 mg/m2 
starting at hr 42 
and continue 
q6hrs, until MTX 
level < 0.1 µM 
 
Repeat MTX 
LCV 1000 mg/m2 
starting at hr 42 
and continue 
q6hrs, until MTX 
level < 0.1 µM 
 
Repeat MTX 
++ If the level is high at hour 36 or 42, but then the patient “catches up” and the level falls to the expected values of 
≤1.0 and/or ≤ 0.4 µM at hours 42 and 48, respectively, resume standard leucovorin and hydration as long as urine 
output remains satisfactory. 
Consider repeat 
MTX level to r/o 
contamination. 
Increase 
hydration while 
awaiting result  
APPENDIX B 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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25 
Suggested post HDMTX infusion guidelines based on standard COG guidelines. 
 
 
 
Concomitant Medication Restrictions: 
1.  Sulfamethoxazole and Trimethoprim (SMX-TPM); synonyms: Bactrim, Co-trimoxazole 
2.  Non-steroidal anti-inflammatory medications (NSAIDS) 
3.  Penicillins 
4.  Proton pump inhibitors 
5.  Aspirin-containing medications 
 
Hold the above medications on the day of HDMTX infusion and for at least 72 hours after the start of the HDMTX 
infusion and until the MTX level is less than 0.4µM (In the presence of delayed clearance continue to hold these 
medications until MTX level is less than 0.1 µM):  
 
 
36 hr MTX 
level 
(if needed)+ 
42 hr MTX 
level 
 
48 hr MTX 
level 
Hydration and leucovorin rescue 
>3 µM 
 
 
 
Increase hydration to 200 mL/m2/hr. 
>10 µM 
 
 
 
Increase hydration to 200 mL/m2/hr. 
 
Consider glucarpidase 
 
≤1.0 µM 
≤ 0.4 µM 
 
No further leucovorin after Hr 54, no further MTX levels after 
Hr 48 
 
1.01-9.9 µM 
0.41-5.9 µM 
 
Continue leucovorin at 15 mg/m2 q 6 hours after Hr 54 until 
plasma MTX is < 0.1 µM 
 
Repeat MTX levels Q 12 hrs 
 
10-19.9 µM 
6-9.9 µM 
 
Leucovorin at 15 mg/m2 at Hr 42 then continue Q 3 hours until 
plasma MTX is < 0.1 µM 
 
Repeat MTX levels Q 12 hrs 
 
Consider glucarpidase 
 
20-200 µM 
10-100 µM 
 
Leucovorin at 100 mg/m2 at Hr 42 then continue q 6 hours until 
plasma MTX is < 0.1 µM 
 
Repeat MTX level Q 12 hrs 
 
Consider glucarpidase 
 
>200 µM 
>100 µM 
 
Leucovorin at 1000 mg/m2 at hr 42 and continue q 6 hours 
until plasma MTX is < 0.1 µM 
 
Repeat MTX level Q 12 hrs 
 
Consider glucarpidase