Clinical trial NCT01178944
Conditions: Adenocarcinoma of the Gastroesophageal Junction Esophageal Undifferentiated Carcinoma Gastric Adenocarcinoma Gastric Squamous Cell Carcinoma Recurrent Esophageal Adenocarcinoma Recurrent Esophageal Squamous Cell Carcinoma Recurrent Gastric Carcinoma Stage IIIB Esophageal Adenocarcinoma Stage IIIB Esophageal Squamous Cell Carcinoma Stage IIIB Gastric Cancer Stage IIIC Esophageal Adenocarcinoma Stage IIIC Esophageal Squamous Cell Carcinoma Stage IIIC Gastric Cancer Stage IV Esophageal Adenocarcinoma Stage IV Esophageal Squamous Cell Carcinoma Stage IV Gastric Cancer Undifferentiated Gastric Carcinoma
Sponsor: Roswell Park Cancer Institute
Start date: 2010-09-01; Completion date: 2015-11-01
Enrollment: 35 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase II trial studies how well pralatrexate and oxaliplatin work in treating patients with esophageal, stomach, or gastroesophageal junction cancer that cannot be removed by surgery or has spread from the primary site (place where it started) to other places in the body. Pralatrexate may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Drugs used in chemotherapy, such as oxaliplatin, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Giving pralatrexate with oxaliplatin may be an effective treatment for esophageal, stomach, or gastroesophageal junction cancer.

Detailed description:
PRIMARY OBJECTIVES:

I. To determine the overall response rate in patients with advanced esophago-gastric cancer (EGC) to combination pralatrexate and oxaliplatin.

SECONDARY OBJECTIVES:

I. To examine the toxicity and tolerability of this regimen. II. To determine the time-to-progression and overall survival using this regimen.

III. To examine whether functionally relevant polymorphisms of genes of the folate metabolism pathway correlate with efficacy and toxicity of pralatrexate.

IV. To examine whether response to pralatrexate can be predicted by micro-ribonucleic acid (microRNA) expression profiling of the epithelial component of the tumor.

OUTLINE:

Patients receive pralatrexate intravenously (IV) over 3-5 minutes and oxaliplatin IV over 2 hours on day 1. Courses repeat every 2 weeks in the absence of disease progression or unacceptable toxicity. Oxaliplatin will be discontinued after 12 courses.

After completion of study treatment, patients are followed up for 30 days and then periodically thereafter for up to 5 years.

Arms: arm 1: Patients receive pralatrexate IV over 3-5 minutes and oxaliplatin IV over 2 hours on day 1. Courses repeat every 2 weeks in the absence of disease progression or unacceptable toxicity. Oxaliplatin will be discontinued after 12 courses.
Interventions: intervention 1: Laboratory Biomarker Analysis intervention 2: Oxaliplatin intervention 3: Pralatrexate
Intervention descriptions: intervention 1: Correlative studies intervention 2: Given IV intervention 3: Given IV

Protocol documents:
Prot_SAP_000.pdf: 
Page 1 of 2
May 6, 2017
Patrick Boland, MD
Patrick.Boland@RoswellPark.org
Dear  Dr. Boland:
On 5/5/2017, the IRB reviewed the following submission:
Type of Submission:
Modification and Continuing Review
Type of Review: 
 Full Board  
 Expedited  
 Exempt  
 Non-Human Research
Title of Study:
Pralatrexate in Combination with Oxaliplatin in Advanced 
Esophagogastric Cancer:  A Phase II Trial with Predictive Molecular 
Correlates
Investigator:
Patrick Boland
IRB ID:
MODCR00000245 / I 169210
Funding:
Name: NATIONAL COMPREHENSIVE CANCER NETWORK
Grant ID:
None
IND, IDE, or HDE:
None
Documents Reviewed:
• I 169210 IB V9 10113.pdf, Category: Drug Attachment;
• I 169210 Oxaliplatin_ Drug information - UpToDate.pdf, Category: 
Drug Attachment;
• I169219 PT. DIRAY.pdf, Category: Recruitment Materials;
• I 169210 PROT AMD 08 CLN 090115.pdf, Category: IRB Protocol;
• 169210 IND MEMO.pdf, Category: Drug Attachment;
•
I 169219 Consent V10 (data migration only)
The IRB approved the study from 5/5/2017to 5/17/2018 inclusive.  Before 5/17/2018 or 
within 30 days of study closure, whichever is earlier, you are to submit a continuing 
review with required explanations. You can submit a continuing review by navigating to 
the active study and clicking Create Modification / CR.
If continuing review approval is not granted on or before 5/17/2018, approval of this 
study expires after that date.
Please be advised that only the IRB approved and stamped consent form can be used 
to enroll subjects.  
The principal investigator is responsible for ensuring that the research complies with all 
applicable regulations.  Any modifications in the research project are subject to 
approval by the Board prior to initiation by the investigator. The Board reserves the 
right to stop the research for violations of regulatory or IRB requirements.
Elm & Carlton Streets
Buffalo, New York 14263
Institutional Review Board
Approval Notice
This institution has an approved assurance of compliance on file with HHS which 
covers this activity FWA 00006731 Federal Wide Assurance identification 
number


Page 2 of 2
A progress report must be submitted to the IRB at least one month prior to the expiration 
date noted above for continuing review as required by federal regulations and/or 
institutional requirements.  
Please be advised that your research study may be audited periodically by the IRB for 
compliance.
This activity has been reviewed and approved by an IRB in accordance with the 
requirements of 45 CFR 46, including its relevant Subparts.  This protocol fulfills, when 
applicable, requirements for certifying FDA status for each investigational new drug or 
device.
The study documents have been submitted to Clinical Research Services (CRS) 
Compliance Office for processing prior to release and protocol implementation.  Please 
contact CRS Compliance for information regarding the protocol implementation release 
date. 
In conducting this protocol you are required to follow the requirements listed in the 
INVESTIGATOR MANUAL (HRP-103), including the reporting of Unanticipated Problems 
and any other Reportable New Information.
Sincerely,
Donald Handley MSc, MBA
Camille P Wicher, PhD, Esq., RN, MSN


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Pralatrexate in combination with oxaliplatin in advanced esophago-
gastric cancer: a phase II trial with predictive molecular correlates
RPCI # I 169210
Coordinating Center:
Roswell Park Cancer Institute 
Elm and Carlton Streets 
Buffalo, NY 14263.
Principal Investigator:
Patrick Boland, MD
Department of Medicine
Roswell Park Cancer Institute
Tel: 716- 845-3099
Fax: 716-845-8935.
e-mail: patrick.boland@roswellpark.org
Network Principal Investigator:
Peter Bushunow, MD
Rochester General Hospital
Funding Organization: National Comprehensive Cancer Network (NCCN).
Initial Version:                03.05.10
Amendment #1                 08.26.10
Amendment #2                 12.19.11
Amendment #3
 01.27.12
Amendment #4
 12.21.12
Amendment #5
 01.14.14
            Amendment #6
 06.23.14
            Amendment #7
 10.10.14
Amendment #8 (PI Change) 09.01.15      
Confidentiality Statement:  Any and all information presented in this document shall be treated as 
confidential and shall remain the exclusive property of the parties mentioned above.  The use of such 
confidential information must be restricted to the recipient for the agreed purpose and must not be disclosed, 
published or otherwise communicated to any unauthorized persons, for any reason, in any form whatsoever 
without the prior written consent of the parties above.
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Network Investigator Signature Page
I 169210
Pralatrexate in combination with oxaliplatin in advanced esophago-gastric cancer: a phase 
II trial with predictive molecular correlates
PROTOCOL APPROVAL AND INVESTIGATOR AGREEMENT
I have read and familiarized myself with this protocol and I agree to conduct the study as 
described according to GCP and ICH guidelines.
Principal Investigator
Signed
Date
Printed
Address:
Phone:
Fax:
E-mail:
NOTE: Please see Appendix 2 for Network-specific instructions that will apply to 
your site.
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STUDY SYNOPSIS:
Objectives:
Primary:  
•
To determine the overall response rate in patients with advanced esophago-gastric cancer 
to combination pralatrexate and oxaliplatin.
Secondary:
•
To examine the toxicity and tolerability of this regimen
•
To determine the time-to-progression and overall survival using this regimen
•
To examine whether functionally relevant polymorphisms of genes of the folate 
metabolism pathway correlate with efficacy and toxicity of pralatrexate
•
To examine whether response to pralatrexate can be predicted by microRNA expression 
profiling of the epithelial component of the tumor.
Design:
Phase II with a safety lead-in cohort of 6 patients to assess safety and tolerability of the 
combination.  Total accrual will be 34 (single institution) and duration will be 4 years with early 
stoppage rules for futility. 
Major Inclusion / Exclusion Criteria:
Inclusion
1. Histologically confirmed carcinoma of the esophagus, stomach or gastro-esophageal 
junction that is metastatic, or locally advanced and inoperable for cure.  Histologic sub-
types permitted include adenocarcinoma, squamous-cell carcinoma, or undifferentiated 
carcinoma.  Small-cell carcinoma variant is not eligible.
2. No previous systemic therapy for metastatic or recurrent disease.  Therapy 
(chemotherapy, radiotherapy, or both) administered in the neo-adjuvant, adjuvant, or 
definitive setting for previously localized disease is permitted, provided it was completed 
more than 6 months prior to enrollment.  Palliative radiotherapy is permitted provided it 
is completed > 3 weeks prior to study therapy initiation.
3. ECOG performance status 0-2.
4. Life expectancy > 12 weeks.
5. Age > 18 years.
6. Adequate hematologic parameters (hemoglobin > 9 g/dl, absolute neutrophil count > 
1500/mm3, platelet count > 100,000/mm3).
7. Adequate biochemical parameters (serum creatinine < institutional upper limit normal 
(ULN), bilirubin < 1.5 X ULN, transaminases < 3 X ULN; for documented liver 
metastases, transaminases up to 5 X ULN is permitted).
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8. No evidence of > grade 2 peripheral neuropathy.
9. Patients with reproductive potential must be willing to use an adequate contraceptive 
method (e.g., abstinence, intrauterine device, oral contraceptives, barrier device with 
spermicide or surgical sterilization) during treatment and for three months after 
completing treatment.  A negative pregnancy test is required for women of child-bearing 
potential.  Nursing women are ineligible.
10. Written, informed consent.
Exclusion
1. Hypersensitivity to platinum compounds.
2. Uncontrolled inter-current illness including but not limited to active infection, 
symptomatic congestive heart failure, unstable angina, uncontrolled cardiac arrhythmia, 
or psychiatric illness that would limit compliance with study requirements.
3. Presence of brain metastases.
4. Patients with third-space (pleural, peritoneal) fluid not controllable with usual drainage 
methods are not eligible.
5. History of second primary malignancy within 3 years prior to enrollment, except for in-
situ cervix carcinoma or non-melanoma skin cancer.
6. Undergone an allogeneic stem cell transplant.
Treatment Plan:
Following Vitamin B12 and folate supplementation, and appropriate anti-emetic therapy (to 
include a 5-HT3 antagonist and a steroid), pralatrexate will be administered intravenously in a 
dose of 120 mg/m2 over 3-5 minutes followed by oxaliplatin at 85 mg/m2 intravenously over 
2 hours.  Cycles will be repeated every 2 weeks until disease progression, undue toxicity, or 
patient/physician discretion.  For patients meeting response criteria to continue therapy, 
oxaliplatin will be discontinued after 12 cycles (maximum cumulative dose = 1020 mg/m2) to 
reduce the risk of peripheral neuropathy.
The safety lead-in will start at cohort 1 with six patients.  If this is tolerable, then all further 
accrual will be at this starting dose level.  If not, successively decreasing cohorts will be 
investigated per the following table: 
Cohort 1
Cohort -1 (if 
needed)
Cohort -2 (if 
needed)
Oxaliplatin
85mg/m2
85mg/m2
65mg/m2
Pralatrexate
120mg/m2
100mg/m2
80mg/m2


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TABLE OF CONTENTS
1. HYPOTHESIS .........................................................................................................................6
2. BACKGROUND......................................................................................................................6
3. RESEARCH DESIGN.............................................................................................................7
4. OBJECTIVES
......................................................................................................................8
5. INCLUSION OF WOMEN AND MINORITIES .................................................................8
6. STUDY DRUGS.......................................................................................................................8
6.1  PRALATREXATE ...........................................................................................................8
6.2  OXALIPLATIN ..............................................................................................................20
7. STUDY POPULATION ........................................................................................................24
8. TREATMENT PLAN............................................................................................................25
9. SUPPORTIVE CARE GUIDELINES .................................................................................27
10. DATA REQUIREMENTS & STUDY CALENDAR..........................................................27
11. SAFETY LEAD-IN COHORT…………………………………………………………….28
12. DOSE DELAYS & MODIFICATIONS…………………………………………………...30
13. END-POINTS……………………………………………………………………………… 36
14. STATISTICAL CONSIDERATIONS .................................................................................36
15. CORRELATIVE STUDIES..................................................................................................37
16. ADVERSE EVENT REPORTING ......................................................................................42
17. RESPONSE EVALUATION ................................................................................................47
18. ETHICAL AND REGULATORY STANDARDS………………………………………..53
19. STUDY RESPONSIBILITIES…………………………………………………………….54
20. MAINTENANCE OF STUDY DOCUMENTS…………………………………………..55
21. ADMINISTRATIVE RULES……………………………………………………………...55
REFERENCES.............................................................................................................................56
APPENDICES………………………………………………………………………………….61
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1. Hypothesis
The efficacy of current chemotherapy options in advanced esophago-gastric cancer has 
reached a plateau.  Investigative strategies should focus on the development of new agents 
and on predictive markers to tailor the efficacy of drugs to specific patient or tumor 
characteristics.  We hypothesize the following: 
a.
The combination of pralatrexate and oxaliplatin will be efficacious and tolerable in 
patients with advanced esophago-gastric cancer.  
b. Polymorphisms in genes responsible for transport and metabolism of pralatrexate will 
correlate with response and outcome.
c.
MicroRNA (miRNA) expression profiles generated using cell line data can predict 
response to pralatrexate using expression profiling of the epithelial component of the 
tumor isolated using laser-capture microdissection (LCM).
2. Background
Cancers of the stomach and esophagus are the second and sixth most common causes of 
cancer-related mortality world-wide.1  With delayed symptoms, most cancers present at 
an advanced stage and the 5-year survival remains less than 20%.  There has been a 
dramatic increase in the incidence of esophageal adenocarcinoma in the United States in 
the past two decades with a concurrent 7-fold increase in mortality.2  Anatomically, most 
cancers of the upper gastrointestinal tract arise from the distal esophagus, gastro-
esophageal junction (GEJ) or the proximal stomach, and are typically of the 
adenocarcinoma sub-type.  There is no uniform global standard for the first-line therapy 
of advanced esophago-gastric cancer (EGC).  Regimens include doublet or triplet- 
combination chemotherapy agents, usually platinum- or fluorouracil-based with response 
rates ranging from 15%-50%, achieving an overall survival of 9-11 months.3-6
Oxaliplatin, a novel antineoplastic platinum is a favorable alternative to cisplatin in terms 
of toxicity without compromise of efficacy.7  It is less emetogenic, less nephrotoxic and 
less neurotoxic compared to cisplatin.  It has been widely incorporated into combination 
treatment regimens in advanced EGC.8-13  Our group has systematically demonstrated the 
feasibility and efficacy of oxaliplatin in combination with 5-fluorouracil (5FU), and with 
capecitabine in three successive trials in esophagus cancer (EC), all in combination with 
radiotherapy.14-16  This regimen showed an impressive pathologic complete response rate 
of 33% for resectable EC in SWOG 0356, a multi-center phase II trial.17  Major 
drawbacks of the oxaliplatin plus 5FU doublets are inconvenience of the 5FU infusion 
schedule and the need for central venous access and infusion pumps.  Hence it is 
imperative to investigate newer agents in combination with platinums.  
The anti-folate drugs methotrexate and pemetrexed have demonstrated clinical activity in 
EGC.18-21  Pemetrexed has shown greater efficacy compared to 5FU against several 
gastric cancer cell lines.22  This drug shows synergistic cytotoxicity when combined with 
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oxaliplatin in human HT29 colon cancer cells, including 5FU-resistant cells.23  Recently, 
a phase II trial assessing   pemetrexed (500mg/m2) in combination with oxaliplatin 
(120mg/m2) every 21 days in advanced gastric cancer demonstrated a response rate of 
36%, with four complete responses and an impressive time-to-progression of 6.2 
months.24  With the more conventionally used biweekly regimen of oxaliplatin at 
85mg/m2, pemetrexed can be safely given every 14 days at a dose of 400-500mg/m2 in 
patients without extensive prior therapy.25  Similar combinations have been investigated 
in non-small cell lung cancer.26  Taken together, these data suggest that the combination 
of oxaliplatin with the multi-targeted anti-folate pemetrexed is safe and efficacious in 
several tumor types, including gastric cancer.
Pre-clinical observations of increased cellular uptake and polyglutamylation of 
pralatrexate relative to other anti-folates methotrexate and pemetrexed make this an 
appropriate drug for study in clinical trials.27-30  It has demonstrated synergy with 
platinum and taxane compounds.29, 31  Phase I testing has evaluated weekly, biweekly and 
every 3-week dosing with stomatitis being the dose-limiting toxicity.   The documented 
pre-clinical synergy with platinum makes it an ideal candidate for testing in a 
combination regimen.  Of note, these trials did not utilize vitamin supplementation to 
mitigate toxicity.  With this approach, higher doses of pralatrexate are currently being 
tested in clinical trials.  In a phase I study, pralatrexate at 120mg/m2 in combination with 
docetaxel at 35mg/m2, both administered every other week was found to be safe and 
tolerable when vitamin supplementation was administered concurrently.31  
Using this information, we propose to use pralatrexate at 120mg/m2 every other week in 
combination with the standard dose of biweekly oxaliplatin (85mg/m2) in this clinical 
trial for patients with advanced EGC.  The lack of overlapping toxicities for these drugs 
makes this particularly attractive.  As there is no phase I data for this specific 
combination, a safety lead-in cohort of 6 patients will be tested first.   
3. Research Design
This phase II study will consist of combination pralatrexate and oxaliplatin every 2 weeks 
in the first-line treatment of patients with advanced EGC following an initial safety-lead 
in cohort.  Response will be assessed every 8 weeks and treatment will continue until 
disease progression.  Oxaliplatin will be discontinued after 12 cycles of treatment to 
reduce the risk of neuropathy with cumulative dosing in patients with responding or 
stable disease.  The primary clinical end-point is response rate with secondary end-points 
of describing toxicity and calculating time-to-progression and overall survival. 
Blood (5ml) will be collected on all patients and sent to the Adjei laboratory for single 
nucleotide polymorphism (SNP) analysis.  In addition, pre-treatment primary tumor 
biopsies will be undertaken by upper endoscopy for LCM for miRNA expression 
profiling.  This will be sent to the Yendamuri laboratory. See correlative section for 
details.
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4. Objectives:
4.1.
Primary
To determine the overall response rate in patients with advanced EGC to combination 
pralatrexate and oxaliplatin.
4.2.
Secondary
    4.2.1.
To examine the toxicity and tolerability of this regimen
    4.2.2.
To determine the time-to-progression and overall survival using this 
regimen
    4.2.3.
To examine whether functionally relevant polymorphisms of genes of the 
folate metabolism pathway correlate with efficacy and toxicity of 
pralatrexate
    4.2.4
To examine whether response to pralatrexate can be predicted by 
microRNA expression profiling of the epithelial component of the tumor.
5. Inclusion of Women and Minorities
Women and minorities will be eligible for this study without any alteration in the 
eligibility criteria.  National demographics for esophagus cancer indicate a relative rarity 
of this disease in women.  Hence only 10-15% of patients enrolled on esophagus cancer 
trials are women.  African-Americans comprise 5-10% of patients on esophagus cancer 
trials with most of these patients developing squamous-cell cancer.  Our own experience 
suggests a similar demographic distribution and the current clinical trial would be 
expected to mirror a similar patient population.  
6. Study Drugs
6.1
PRALATREXATE
PHARMACOLOGY
Pralatrexate is a synthetic 10-deazaaminopterin antifolate. The 10-deazaaminopterins are 
a class of folate analogues that demonstrate greater antitumor effects than methotrexate 
against human tumor cell lines and in human tumor xenograft models in mice.27, 28, 32  
Pralatrexate is a folate analogue that inhibits folate metabolism by binding to and 
inhibiting the enzyme dihydrofolate reductase (DHFR). The improved antitumor effects 
of pralatrexate are likely due to the more effective internalization via reduced folate 
transport, and the subsequent accumulation in tumor cells through the formation of 
polyglutamylated metabolites.28, 33
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PHYSICAL, CHEMICAL, PHARMACEUTICAL PROPERTIES
Pralatrexate has the chemical name (2S)-2-[[4-[(1RS)-1-[(2, 4-diaminopteridin-6-
yl)methyl]but-3-ynyl]benzoyl]amino]pentanedioic acid.  The structural formula is as 
follows:
Pralatrexate is a 1:1 racemic mixture of S- and R- diastereomers at the C10 position 
(indicated with *)
The molecular formula is C23H23N7O5 and the molecular weight is 477.48 g/mol. 
Pralatrexate is an off-white to yellow solid.  It is soluble in aqueous solutions at pH 6.5 or 
higher.  Pralatrexate is practically insoluble in unbuffered water, chloroform, and ethanol.  
The pKa values are 3.25, 4.76, and 6.17. 
Pralatrexate is supplied as a preservative free, sterile, isotonic, non-pyrogenic clear 
yellow aqueous parenteral solution contained in a single-use 2 mL-size clear glass vial 
for intravenous administration.  Each 1 mL of solution contains 20 mg of pralatrexate, 
0.6% sodium chloride to achieve an isotonic (280-300 mOsm) solution, and sufficient 
sodium hydroxide, and hydrochloric acid if needed, to adjust and maintain the pH at 
7.5-8.5.  Pralatrexate is supplied as either 20 mg (1 mL) or 40 mg (2 mL) single-use vials 
at a concentration of 20 mg/mL.
MECHANISM OF ACTION
Pralatrexate is a folate analog metabolic inhibitor that competitively inhibits DHFR.  In in 
vitro studies, pralatrexate showed improved cytotoxic activity against a panel of cancer 
cell lines compared with methotrexate.  The improved cytotoxic activity is likely due to 
pralatrexate being a more efficient permeant for reduced folate transport and it being 
more efficiently polyglutamylated by the enzyme folylpolyglutamyl synthetase 
(FPGS).28, 33 Similar to what has been reported for methotrexate, cellular uptake of 
pralatrexate is thought to occur via the reduced folate carrier-1 (RFC-1) protein.  RFC-1 
is a membrane protein involved in cellular uptake of reduced folates.  This carrier protein 
has evolved to efficiently transport reduced natural folates into highly proliferative cells 
in order to meet the demands for purine and pyrimidine nucleotides during cell 
replication.  Inside the cell, pralatrexate is polyglutamylated by FPGS, an enzyme 
involved in polyglutamylation of reduced natural folates.  Therefore, pralatrexate may act 
as a competitive inhibitor for polyglutamylation of natural folates.  Further, it is believed 
N
N
N
N
N
H
CO2H
H
NH2
H2N
O
H
*
and epimer at C*
CO2H
CH
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that the addition of glutamate residues to pralatrexate leads to increased intracellular half-
life, thus allowing for prolonged drug action in malignant cells. 
Folic acid (folate) is a B vitamin (B9) and serves as a carrier of 1-carbon groups in many 
metabolic reactions that are critical to the life cycle of cells.  Following absorption into 
the cell via RFC-1, dietary folate undergoes reduction to dihydrofolate.  Dihydrofolate is 
then converted to tetrahydrofolate (THF) by the enzyme DHFR.  THF is required for the 
synthesis and catabolism of several amino acids, formation of creatine and choline, 
synthesis of purines, methylation of ribonucleic acids (RNAs), and synthesis of 
thymidine.  Pralatrexate is a competitive inhibitor of DHFR, and thus treatment with 
pralatrexate will lead to depletion of thymidine and other biological molecules the 
synthesis of which depends on one carbon transfer.  Depletion of thymidine will result in 
inhibition of DNA replication and thus interference with cell proliferation.  For detailed 
information about the general pharmacology, pharmacokinetics (PK), nonclinical 
toxicology and clinical efficacy studies of pralatrexate, refer to the current pralatrexate 
Investigator’s Brochure (IB) accompanying this protocol.  
PRECLINICAL INFORMATION
Data from in vitro and in vivo models of human NHL established the superior therapeutic 
activity of pralatrexate compared to methotrexate. In vitro experiments demonstrated that 
pralatrexate concentrations that result in 50% growth inhibition (IC50) were consistently 
10-fold lower than that typically seen for methotrexate in a library of lymphoma cell 
lines, and xenograft studies of lymphoma lines in mice consistently showed pralatrexate 
to be superior to methotrexate.34
Studies exploring the integration of pralatrexate with gemcitabine revealed that these 2 
agents appear to be synergistic in models of lymphoma. One of the important 
observations from these studies was the importance of schedule dependency.35  These 
observations were supported by results from in vitro cytotoxicity experiments, apoptosis 
assays, as well as in vivo xenograft experiments. The sequence of pralatrexate followed 
by gemcitabine was 5 times more effective at inducing apoptosis compared with the 
simultaneous exposure of pralatrexate plus gemcitabine, and in vivo, complete 
regressions were only observed in animals receiving pralatrexate followed by 
gemcitabine.
In cytotoxicity assays using a number of human NSCLC and breast cancer cell lines, 
pralatrexate was found to be superior to both methotrexate (13- to 40-fold lower IC50) and 
another methotrexate analog, edatrexate (2- to 4-fold lower IC50).36
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Table 1:
In Vitro Comparison of Methotrexate Analogs
     Pralatrexate
      Edatrexate
     Methotrexate
DHFR Inhibition, Ki (pM)
13.4
5.8
4.9
FPGS Activity, Vmax/Km
23.2
10.3
2.2
In vitro Activity
IC50 Concentration (μM):
MDA-468 (breast)
0.11
0.39
4.5
SK-BR III (breast)
0.28
0.99
4.2
ZR-75-1 (breast)
0.26
0.86
3.5
SK-LC8 (NSCLC)
0.42
1.24
10.3
SK-LC16 (NSCLC)
0.11
0.26
2.1
Ki = inhibition constant
Vmax = maximum velocity of a reaction catalyzed by a fixed enzyme 
concentration
Km = concentration of substrate which gives ½ Vmax
IC50 = concentration of compound that inhibits cell growth by 50%
Screening of pralatrexate in the National Cancer Institute (NCI) 60-Cell Panel showed 
potent cytotoxic activity across a broad spectrum of tumor types. (Allos data on file)
CLINICAL STUDIES: HEMATOLOGIC MALIGNANCIES
Study PDX 02-078
In a “phase 2/1/2” study of pralatrexate in 57 patients with relapsed or refractory 
lymphomas, the initial phase 2 dose of 135 mg/m2 every 2 weeks was associated with 
unacceptable mucositis, leading to phase 1/2 evaluation of 30 mg/m2 once weekly for 6 
weeks in 7-week cycles. This schedule modification resulted in a 50% reduction in the 
major hematologic toxicities and abrogation of the grade 3 to 4 stomatitis. Overall 
response rate among assessable patients with PTCL was 54% (14/26), including 31% 
(8/26) with complete remission or complete remission unconfirmed, and 23% (6/26) with 
partial remission.37, 38
Study PDX-008
The pivotal clinical trial of pralatrexate for the indication of PTCL was a single-arm 
phase 2 clinical study (PROPEL) in which 115 patients with relapsed or refractory PTCL 
were enrolled. Patients received Pralatrexate 30 mg/m2 once weekly for 6 weeks in 7-
week cycles, and were treated until disease progression or unacceptable toxicity.39, 40  Of 
the 111 patients treated with pralatrexate, 109 were histologically confirmed and 
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evaluable for efficacy. Efficacy was evaluated with IWC by independent central review 
at the end of cycle 1 and then every other cycle thereafter. The primary efficacy endpoint 
was overall response rate and the key secondary efficacy endpoint was duration of 
response until disease progression or death.
Seventy-five patients (68%) were male 34 (32%) were female. Most patients (72%) were 
White and other racial origins included: Black (12%), Hispanic (8%), Asian (6%), other 
and unknown (<1% each).  Patients had an Eastern Cooperative Oncology Group 
(ECOG) performance status at study entry of 0 (39%), 1 (45%), or 2 (17%).  The median 
time from initial diagnosis to study entry was 15.6 months (range 0.8 – 322.3).
The median number of prior systemic therapies was 3 (range 1-12).  Approximately one-
fourth of patients (24%, n = 26) did not have evidence of response to any previous 
therapy.  Approximately two-thirds of patients (63%, n = 69) did not have evidence of 
response to their most recent prior therapy before entering the study.
The overall response rate per IWC by independent central review was 29% (n = 32). 
Overall response rate by investigator assessment was 39% (n = 43). Of the responders, 
63% responded by the first assessment at the end of Cycle 1. The median time to first 
response was 45 days (range, 37–349 days) and the median time to best response was 96 
days (range, 37–483 days). The Kaplan-Meier estimates of the median duration of 
response and overall survival were 10.1 months (range, 1-673 days) and 14.5 months 
(95% CI: 11.2, not evaluable), respectively. 
The mean duration of treatment was 112 days (range, 1–558 days). Most patients (69%, 
n = 77) remained at the target dose (30 mg/m2 for 6 of 7 weeks per cycle) for the duration 
of treatment and 85% of scheduled doses were administered. The most frequent adverse 
events in PROPEL, regardless of causality, were mucositis (71% All Grade, 18% Grade 
3, 4% Grade 4), thrombocytopenia (41% All Grade, 14% Grade 3, 19% Grade 4), nausea 
(41% All Grade, 4% Grade 3, 0 Grade 4), fatigue (36% All Grade, 5% Grade 3, 2% 
Grade 4), anemia (34% All Grade, 16% Grade 3, 2% Grade 4), constipation (34% All 
Grade, 0 Grade 3, 0 Grade 4), pyrexia (34% All Grade, 1% Grade 3, 1% Grade 4), and 
edema (31% All Grade, 1% Grade 3, 0 Grade 4). 
There was an additional analysis of treatment responses among patients in the PROPEL 
trial who had refractory PTCL.40  Refractory disease was defined as 1) no evidence of 
response to their most recent therapy or 2) no evidence of response to any prior therapies. 
Of the 111 patients evaluated for safety, 109 were evaluable for efficacy. Sixty-nine of 
these patients (63%) had no response to their most recent therapy and 26/109 (24%) had 
no response to any prior therapy.  Prior therapies consisted mostly of CHOP-based 
chemotherapy (70%), platinum-containing combination chemotherapy (40%), and non-
platinum containing combination chemotherapy (38%). Of the 69 patients with no 
response to their most recent therapy, the median number of prior systemic therapies was 
3 (range 1-11). There were 18 patients with prior autologous stem cell transplants. 
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The ORR in the 69 patients with no response to their most recent therapy was 25% 
(17/69) based on central review and 36% (25/69) based on investigator review. 
According to central review, the response duration ranged from 41-673 days. In the 26 
patients with no response to any prior therapy, 19% (5/26) had a response to pralatrexate 
according to central review and 27% (7/26) had a response based on investigator review 
(duration of response based on central review, 54-306 days). Of the 69 patients with no 
response to most recent therapy, 46 (67%) received full-dose therapy and the safety and 
tolerability in this subset was similar to the overall study population.
The safety and management of pralatrexate treatment in relapsed or refractory PTCL 
patients included in the PDX-008 was evaluated over 3 parameters: to assess safety 
according to duration of pralatrexate treatment, to evaluate both early- and late- onset 
pralatrexate toxicities, and to assess the impact of pralatrexate dose modification on 
toxicities.41  Patients received pralatrexate 30 mg/m2 administered as an intravenous push 
over 3-5 minutes once weekly for 6 weeks in 7-week cycles. All patients received 
vitamin B12 (1 mg) intramuscular injection every 8-10 weeks and oral folic acid (1.0-1.25 
mg) daily. Patients with methylmalonic acid (MMA) levels ≥ 200 nmol/L or 
homocysteine (Hcy) ≥ 10 μmol/L at screening received supplementation > 10 days prior 
to the first pralatrexate dose.  Pralatrexate doses were omitted in patients with platelets 
<50,000/μL, absolute neutrophil count (ANC) 500-1,000/μL with fever, ANC <500 μL, 
Grade 2-4 mucositis and any other Grade 3 treatment-related event. The dose was 
reduced to 20 mg/m2 in patients with platelets < 25,000/μL on 2 occurrences, recurrence 
of ANC 500-1,000μL with fever, recurrence of ANC <500μL, recurrence of Grade 2 
mucositis, Grade 3-4 mucositis, or any Grade 4 treatment-related event.  Of the 111 
patients evaluated for safety, 109 were evaluable for efficacy. Patients received 
pralatrexate for a median of 70 days (range 1-696); 19 of these patients received therapy 
for > 180 days; and 12 received therapy for ≥ 300 days. Two or more cycles were 
delivered in 64 patients, and ≥ 3 cycles were delivered in 43 patients. The median 
cumulative dose was 207.9 mg/m2 (range 26.7-2108.8). There were 76 patients (68%) 
who needed no dose modification. Of the remaining 35 patients (32%), dose reduction 
occurred in cycle 1 for 20 patients, 8 in cycle 2, and 7 in cycle 3 or later. The most 
common reason for dose reduction was mucosal inflammation. Prior to dose 
modification, the frequency of Grade 2-4 mucosal inflammation was 28/35 compared 
with 15/35 after dose modification. 
The most common adverse events in the overall population were mucosal inflammation, 
nausea, thrombocytopenia, and fatigue. There was no evidence of cumulative dose-
toxicity effects. 
A additional analysis evaluated the correlation between baseline methylomalonic acid 
(MMA) status and mucositis severity in the PROPEL study, and the implications for 
vitamin prophylaxis.42  In addition to MMA, homocysteine (HCY) and red blood cell 
(RBC) folate levels were evaluated to assess the association between these baseline 
parameters and mucositis and thrombocytopenia.  Mucositis and thrombocytopenia were 
the most common adverse events reported in the PROPEL study. Of the 111 patients 
evaluated for safety, 109 were evaluable for efficacy. The following percentages of 
patients had baseline levels available: MMA 89%, HCY 91%, RBC folate 75%. 
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There was a statistically significant linear relationship between mucositis grade and 
baseline MMA level. (slope estimate = 43.3 nmol/L, P=0.039). In the subset of patients 
enrolled in PROPEL for whom baseline MMA levels were obtained, those with higher 
levels of baseline MMA compared to the normal range  experienced increased severity of 
mucosal inflammation despite concurrent vitamin supplementation. Although not 
significant, there was a trend toward a relationship between increasing severity of 
thrombocytopenia and increasing MMA levels (slope estimate= 17.6 nmol/L, P=0.267). 
There were no significant relationships between baseline HCY and RBC folate levels and 
mucosal inflammation or thrombocytopenia. 
Patients with higher baseline levels of MMA experienced increased severity of mucositis. 
Based on the results of this study, vitamin supplementation with folic acid and vitamin 
B12 is appropriate in patients with relapsed or refractory PTCL who are treated with 
pralatrexate.
Study PDX-010
An open-label phase 1 trial was conducted to examine the safety and efficacy of 
pralatrexate in relapsed or refractory cutaneous T-cell lymphoma (CTCL).  Results from 
31 enrolled patients were presented at the American Society of Hematology 2009 Annual 
Meeting.43  Eligible patients were required to have mycosis fungoides stage 1B or higher 
(MF), Sezary syndrome (SS), or primary cutaneous anaplastic large cell lymphoma 
(ALCL), and progression or relapse of disease after at least 1 systemic therapy and a 
ECOG Performance Status of ≤ 2. The 31 evaluable patients (22 with MF, 7 with SS, 1 
with primary cutaneous ALCL, 1 with CTCL, NOS) were enrolled into 6 cohorts: 
pralatrexate 30 mg/m2 for 3 of 4 weeks (n = 2), 20 mg/m2 for 3 of 4 weeks (n = 3), 20 
mg/m2 for 2 of 3 weeks (n = 7), 15 mg/m2 for 3 of 4 weeks (n = 6), 15 mg/m2 for 2 of 3 
weeks (n = 3), 10 mg/m2 for 3 of 4 weeks (n = 10). All patients received vitamin B12 and 
folic acid supplementation. These patients were heavily pretreated with a median of 6 
prior regimens (range, 1–25), and a median of 4 prior systemic regimens (range, 1–10).
Of the 31 evaluable patients, 12 achieved a response (39%), including complete response 
in 2 patients (1 each with MF/SS and primary cutaneous ALCL) and partial response in 
10 patients (all with MF/SS). The ORR at pralatrexate dose intensity ≥ 15 mg/m3 q3/4 
week was 61% (11/18). A pralatrexate dose of ≥ 15 mg/m3 seemed to be the threshold for 
substantial activity in CTCL and the optimal regimen was pralatrexate ≥ 15 mg/m3  
weekly 3 of 4 week cycle. Dose-limiting toxicities (DLTs) included Grade 2 anorexia 
(n=1), Grade 3 weakness (n=1), Grade 2 stomatitis (n= 3), Grade 2-3 stomatitis (n=1), 
Grade 3 LFT abnormalities (n=1), Grade 2 fatigue (n=1), Grade 2 dehydration (n=1), 
Grade 3 thrombocytopenia/neutropenia (n=1), Grade 3 skin lesion (n=1), and Grade 3 
zoster (n=1).  The most common treatment-related adverse events included stomatitis 
(Grade 1-3= 58%, Grade 3= 13%), nausea (Grade 1-3= 52%, Grade 3 = 0%), and fatigue 
(Grade 1-3= 48%, Grade 3 = 0%).
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Pralatrexate shows activity in patients with relapsed /refractory CTCL (median of 4 prior 
systemic therapies) at a lower dose-intensity than in studies for PTCL. An expansion 
cohort is enrolling additional patients at pralatrexate 15 mg/m2 weekly 3 of 4 week cycle. 
Study PDX-009
It was demonstrated in non-Hodgkin’s lymphoma (NHL) cell lines in vitro and 
xenografts in vivo that pralatrexate may synergize with gemcitabine in a schedule-
dependent manner.35  pralatrexate in combination with gemcitabine is being evaluated in 
an ongoing clinical study (Study PDX-009) in patients with certain relapsed or refractory 
lymphoproliferative malignancies, including Hodgkin’s lymphoma and certain types of 
NHL (www.clinicaltrials.gov/show/NCT00481871). (Data on file, Allos Therapeutics) 
The phase 1 component of the study has been completed.44  Various doses and schedules 
were evaluated in the following treatment groups:
•
Treatment Group A (n=7): pralatrexate (15 mg/m2) followed the next day by 
gemcitabine (400 mg/m2-starting dose), administered once weekly. One cycle of 
pralatrexate and gemcitabine was 4 weeks in duration consisting of 3 weeks of treatment 
followed by 1 week of rest.  Accrual was closed in this treatment group due to dose 
limiting toxicities (DLT) of Grade 3-4 neutropenia and thrombocytopenia.
•
Treatment Group B (n=10): pralatrexate (10 mg/m2) followed the next day by 
gemcitabine (300 mg/m2- starting dose), administered once every 2 weeks. One cycle of 
pralatrexate and gemcitabine is 4 weeks in duration consisting of 2 doses each of 
pralatrexate and gemcitabine.
•
Treatment Group C (n=17): pralatrexate (10 mg/m2) followed 1 hour later by 
gemcitabine (300 mg/m2- starting dose), administered once every 2 weeks. One cycle of 
pralatrexate and gemcitabine is 4 weeks in duration consisting of 2 doses each of 
pralatrexate and gemcitabine.
Patients were enrolled into each treatment group until the maximum tolerated dose 
(MTD) was determined. The MTD was defined as the highest dose level at which ≤ 33% 
of patients experience a DLT. Potential DLTs in the phase 1 portion of the study 
included: Grade 4 neutropenia for ≥ 7 days, Grade 4 thrombocytopenia or any 
thrombocytopenia with clinically significant bleeding, ≥ Grade 3 febrile neutropenia, ≥ 
Grade 3 treatment-related nonhematological toxicity excluding nausea/vomiting in the 
absence of appropriate antiemetic therapy. Safety information was obtained for all 
patients, and patients who received ≥ 1 cycle of chemotherapy were eligible for the 
efficacy evaluation.  Of the 35 patients treated in the phase 1 component, 33 were 
evaluable for response. The median number of prior regimens among the 35 patients was 
3 (range 1-11). Lymphoma subtypes included: B-cell (n=15 [DLBCL= 11, follicular-4]), 
PTCL (n=12), Hodgkin’s lymphoma (n=7), composite B- and T-cell lymphoma (n=1).   
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The MTD in Groups B and C were pralatrexate/gemcitabine 10/400 mg/m2, when given 
on sequential days, and pralatrexate/gemcitabine 15/600 mg/m2, when given on the same 
day. Grade 3-4 treatment-related adverse events (with either study drug) occurring in > 2 
patients included anemia (29% Grade 3, 0% Grade 4), neutropenia (31% Grade 3, 11% 
Grade 4 ), thrombocytopenia (11% Grade 3 , 26% Grade 4 ), leukopenia (3% Grade 3, 
9% Grade 4 ), and abnormal liver function test (9% Grade 3 , 0% Grade 4 ). In Group B, 
33% of patients had DLTs (Grade 4 thrombocytopenia or any thrombocytopenia with 
clinically significant bleeding 17%, ≥ Grade 3 febrile neutropenia 17%, ≥ Grade 3 
treatment-related nonhematological toxicity 33%). In Group C, 21% of patients had 
DLTs (Grade 4 thrombocytopenia or any thrombocytopenia with clinically significant 
bleeding 7%, ≥ Grade 3 treatment-related nonhematological toxicity 14%). 
Response to therapy was evaluated using the International Workshop Criteria a minimum 
of every 3 cycles. Of the 33 evaluable patients, 8 (24%) had a partial response 
(Hodgkin’s lymphoma 4/7, PTCL 2/11, B-cell lymphoma 2*/15 [*1 patient with mixed 
B- and T-cell histology]). The authors concluded that the combination of pralatrexate and 
gemcitabine can be administered on a q2w schedule with acceptable toxicity. Based on 
the results of the study, the phase 2 component is actively accruing using the MTD in 
Groups B (pralatrexate [10 mg/m2] followed the next day by gemcitabine [300 mg/m2- 
starting dose], administered once every 2 weeks, one cycle is 4 weeks duration) and C 
(pralatrexate [10 mg/m2] followed 1 hour later by gemcitabine [300 mg/m2- starting 
dose], administered once every 2 weeks, one cycle is 4 weeks duration) of the phase 1 
component.
The phase 2 expansions at the MTD will evaluate both sequential dosing (pralatrexate 10 
mg/m2 and gemcitabine 400 mg/m2) and same-day dosing (pralatrexate 15 mg/m2 and 
gemcitabine 600 mg/m2) in a q2 week schedule.
CLINICAL STUDIES: SOLID TUMORS (Non-Small Cell Lung Cancer)
Study 97-006
The first Phase 1 study was conducted exclusively in patients with NSCLC, who had 
been previously treated with a median of 2 prior chemotherapy regimens. Initially, 
pralatrexate was administered to 6 patients at a dose of 30 mg/m2 IV weekly for 3 weeks 
out of 4 weeks. Due to the excessive incidence of mucositis/stomatitis/pharyngitis 
(mucositis) in these patients, which became the dose-limiting toxicity (DLT), the dosing 
schedule for the remaining patients was changed to a once every 2 week treatment on a 
28-day cycle, starting at 15 mg/m2 q 2 weeks and escalated to a maximum dose of 170 
mg/m2 q 2 weeks. Based on these Phase 1 data, the recommended dose for subsequent 
Phase 2 studies was 150 mg/m2 administered q 2 weeks.45
Study 99-053
A subsequent single-agent Phase 2 study, in patients with Stage IIIB or IV NSCLC 
revealed activity of pralatrexate consistent with the activity of other single agents in this 
setting. Of 38 evaluable patients, 4 patients (11%) experienced a confirmed partial 
response (PR) lasting 4, 9, 12, and 15 months respectively, while 12 patients (31%) 
exhibited stabilization of their disease (SD).46 
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Study PDX-012
An exploratory Phase 2 trial in NSCLC has recently completed enrollment. The goal of 
this study is evaluate the effectiveness and safety of pralatrexate monotherapy at a 
starting dose of 190 mg/m2 every 2 weeks compared to erlotinib when given to NSCLC 
patients who are current or former cigarette smokers and who have received at least 1 
prior treatment with a platinum drug (cisplatin or carboplatin).
SAFETY OVERVIEW
The most common adverse events observed in patients with peripheral t-cell lymphoma 
(PTCL) treated with pralatrexate were mucositis, thrombocytopenia, nausea, and fatigue. 
Patients receiving pralatrexate should be monitored closely because AEs may occur at 
any time during therapy. For detailed information about the safety profile of pralatrexate, 
refer to the current pralatrexate IB.
 
Clinical Trials Experience
The safety of pralatrexate was evaluated in 111 PTCL patients in a single-arm clinical 
study (PDX-008) in which patients received 30 mg/m2 once weekly for 6 weeks in 7-
week cycles.  The median duration of treatment was 70 days (range 1-540 days). 
Most Frequent Adverse Events
Table 1 summarizes the most frequent adverse events in PDX-008, regardless of 
causality, using the National Cancer Institute-Common Terminology Criteria for Adverse 
Events (NCI CTCAE, version 3.0).


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Table 2: Adverse Reactions Occurring in PTCL Patients (Incidence ≥ 10% of 
patients) 
PDX-008
(n=111)
Total
Grade 3
Grade 4
    Preferred Term
n
(%)
n
(%)
n
(%)
Any Adverse Event
111
(100)
48
(43)
34
(31)
     Mucositisa 
78
(70)
19
(17)
4
(4)
     Thrombocytopeniab 
45
(41)
15
(14)
21
(19)b
     Nausea
44
(40)
4
(4)
0
(0)
     Fatigue
40
(36)
5
(5)
2
(2)
     Anemia 
38
(34)
17
(15)
2
(2)
     Constipation
37
(33)
0
(0)
0
(0)
     Pyrexia
36
(32)
1
(1)
1
(1)
     Edema 
33
(30)
1
(1)
0
(0)
     Cough
31
(28)
1
(1)
0
(0)
     Epistaxis
29
(26)
0
(0)
0
(0)
     Vomiting
28
(25)
2
(2)
0
(0)
     Neutropenia 
27
(24)
14
(13)
8
(7)
     Diarrhea
23
(21)
2
(2)
0
(0)
     Dyspnea
21
(19)
8
(7)
0
(0)
     Anorexia 
17
(15)
3
(3)
0
(0)
     Hypokalemia 
17
(15)
4
(4)
1
(1)
     Rash
17
(15)
0
(0)
0
(0)
     Pruritus 
16
(14)
2
(2)
0
(0)
     Pharyngolaryngeal pain
15
(14)
1
(1)
0
(0)
     Liver function test abnormalc
14
(13)
6
(5)
0
(0)
     Abdominal pain
13
(12)
4
(4)
0
(0)
     Pain in extremity
13
(12)
0
(0)
0
(0)
     Back pain
12
(11)
3
(3)
0
(0)
     Leukopenia 
12
(11)
3
(3)
4
(4)
     Night sweats
12
(11)
0
(0)
0
(0)
     Asthenia
11
(10)
1
(1)
0
(0)
     Tachycardia
11
(10)
0
(0)
0
(0)
     Upper respiratory tract infection
11
(10)
1
(1)
0
(0)
a Stomatitis or Mucosal Inflammation of the gastrointestinal and genitourinary tracts.
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bFive patients with platelets < 10,000/μL
cAlanine Aminotransferase, Aspartate Aminotransferase, and Transaminases Increased
HANDLING AND STORAGE OF PRALATREXATE
Pralatrexate Injection Formulation 
Pralatrexate Injection will be supplied by Allos and will have been tested and released 
according to established specifications. Pralatrexate Injection is formulated as a sterile 
solution for injection and will be supplied in single-use clear glass vials containing 
pralatrexate at a concentration of 20 mg/mL as a preservative-free, sterile, clear yellow 
solution individually packaged for intravenous use.
Pralatrexate Injection Storage and Handling 
Pralatrexate Injection is a cytotoxic agent. The institutional, local, and all applicable 
policies and procedures must be followed for proper handling and disposal of 
chemotherapy drugs.
 
Pralatrexate Injection must be stored refrigerated at 2-8°C (36-46°F) and protected from 
light. Pralatrexate vials should be stored in original carton until use. Unopened vial(s) of 
Pralatrexate are stable if stored in the original carton at room temperature for 72 hours.  
Any vials left at room temperature for greater than 72 hours should be discarded.
Preparation and Administration Precautions
Pralatrexate is a cytotoxic anticancer agent. Caution should be exercised in handling, 
preparing, and administering of the solution.  The use of gloves and other protective 
clothing is recommended.  If Pralatrexate comes in contact with the skin, immediately 
and thoroughly wash with soap and water.  If Pralatrexate comes in contact with mucous 
membranes, flush thoroughly with water.  
PREPARATION FOR INTRAVENOUS PUSH ADMINISTRATION
1.
Pralatrexate vials should be refrigerated at 2-8°C (36-46°F) until use.
2.
Pralatrexate vials should be stored in original carton to protect from light until 
use.
3.
Pralatrexate is a clear, yellow solution.  Parenteral drug products should be 
inspected visually for particulate matter and discoloration prior to administration, 
whenever solution and container permit.  Do not use any vials exhibiting 
particulate matter or discoloration.  
4.
The calculated dose of Pralatrexate should be aseptically withdrawn into a syringe 
for immediate use.
5.
Do not dilute Pralatrexate.
6.
Administer Pralatrexate by IV push over 3 to 5 minutes via the side port of a free 
flowing 0.9% Sodium Chloride Injection, USP IV line.
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7.
Pralatrexate vials contain no preservatives and are intended for single use only.  
After withdrawal of dose, discard vial including any unused portion. 
8.
Unopened vial(s) of Pralatrexate are stable if stored in the original carton at room 
temperature for 72 hours.  Any vials left at room temperature for greater than 72 
hours should be discarded.
6.2
OXALIPLATIN
Chemical Name:
cis-[(1 R,2R)-1,2-cyclohexanediamine-N,N’]
[oxalate(2-)-O,O’]platinum.
International Non-Proprietary Name: EloxatinTM
Empirical Formula:
C8H14N2O4Pt
Relative Molecular Weight:
397.3
Physical Properties:
Slightly soluble in water
Very slightly soluble in methanol
Practically insoluble in ethanol and acetone
Structural Formula:
Oxaliplatin is a diaminocyclohexane (DACH) - platinum compound, active in several 
solid tumor types, including some cisplatin/carboplatin refractory diseases, and licensed 
in the U.S. for the treatment of colorectal cancer. The main mechanism of action of 
oxaliplatin, like cisplatin, is mediated through the formation of DNA-adducts, but 
DACH-platinum adducts are bulkier and more hydrophobic than cisplatin adducts.  
Oxaliplatin as a single agent has a broad spectrum of in vitro cytotoxic/antiproliferative 
activity against a variety of murine and human tumor cell lines.  Oxaliplatin was also 
effective in cell lines with acquired cisplatin resistance.  
Neurotoxicity is considered the dose-limiting toxicity of oxaliplatin. The most common 
acute side effect is a transient peripheral neurotoxicity characterized by paresthesias and 
dysesthesias in hands, feet and the peri-oral area, triggered and/or enhanced by contact 
with cold substances or ambient temperatures. Some patients report laryngo-pharyngeal 
dysesthesia when swallowing cold food or beverages. The intensity of symptoms is 
generally mild to moderate. These symptoms are often observed during an oxaliplatin 
infusion, may last for a few minutes to a few days, and are fully reversible. This acute 
toxicity, which may be observed at doses as low as (or, occasionally, lower than) 90 
mg/m², increases in incidence at higher doses, to affect 75% of patients treated at 200 
mg/m².  Chronic neurotoxicity, by contrast, is a function of cumulative oxaliplatin dose, 
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with a median time of evolution to grade 3 of 23 weeks, but a high rate of reversibility 
once oxaliplatin therapy is discontinued. Women seem more likely than men to 
experience severe neurotoxicity. These symptoms disappear within 12 weeks of stopping 
treatment in 50% of patients and in the majority after 30 weeks.
At doses higher than 45 mg/m², oxaliplatin induces nausea and vomiting with rapid onset 
in the great majority of patients. This can last for 24 to 48 hours and is generally 
controlled by the standard anti-emetic measures used for all platinum derivatives. 
Diarrhea may occur in up to 25% of cycles; when administered with 5-FU/LV, grade 3/4 
diarrhea has been reported in 12% of patients with previously untreated advanced 
colorectal carcinoma. Hematological toxicity is minor and sporadic. In monotherapy 
studies, 2% of patients experienced grade 3-4 anemia and neutropenia; grade 3/4 
thrombocytopenia may also be seen in a small percentage of patients. No significant renal 
toxicity and <1% ototoxicity were reported with oxaliplatin. A single agent, 
pharmacokinetic study of oxaliplatin in patients with renal impairment was performed, 
and demonstrated that single agent oxaliplatin was well tolerated, even in patients with a 
creatinine clearances as low as 20 mg/mL/min. Allergic reactions to Oxaliplatin may 
occur in up to 11% of patients, but few are severe (<0.5% anaphylactic), while fever 
without infection may occur in up to a third of the patients.
Mechanism of Action 
Like cisplatin, oxaliplatin reacts with DNA, forming mainly platinated intrastrand links 
with two adjacent guanines or a guanine adjacent to an adenine.  However, DACH-
platinum adducts formed by oxaliplatin are apparently more effective at inhibiting DNA 
synthesis and are more cytotoxic than cis-diammine-platinum adducts formed from 
cisplatin and carboplatin (21).  The two mechanisms that reproducibly discriminate 
between cisplatin and oxaliplatin are defects in mismatch repair and enhanced replicative 
bypass (22).  Several studies have demonstrated that the loss of the mismatch repair 
(MMR) enzyme complex is a contributor to intrinsic resistance to cisplatin, but not 
oxaliplatin (23).  This resistance can be overcome by a platinum agent with a bulky 
carrier group, such as oxaliplatin, which can make bypass more difficult and can prevent 
the binding of the MMR complex.
Oxaliplatin in Colorectal Cancer
The efficacy of oxaliplatin monotherapy in patients with advanced colorectal cancer was 
evaluated in five phase II trials, two with 63 previously untreated patients and three with 
139 patients with metastatic disease previously treated with and mostly refractory to 5-
FU.  The objective response rate achieved with oxaliplatin as first-line therapy averaged 
18% while that of oxaliplatin as second-line therapy averaged 10%.  These overall 
response rates compare favorably with those of cisplatin (3%) and carboplatin (2.4%) 
resulting from single-agent therapy in patients with metastatic colorectal cancer.  In 
addition, these results were achieved with only moderate toxicity. The efficacy of 
oxaliplatin combined with 5-FU/LV has been evaluated in 614 previously untreated 
patients with metastatic colorectal cancer:  136 patients in two-phase II trials and 478 
patients in three phase III trials.  Response rates varied from 29% to 67%.  Median 
progression-free survival and overall survival ranged from 7.7 to 11 months and 12+ to 
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19 months, respectively.  Major toxicities were diarrhea, stomatitis, peripheral sensory 
neuropathy, nausea and vomiting using oxaliplatin/5-FU/LV combination therapy.47
Intergroup N9741 was a large multicenter randomized phase III study conducted in the 
U.S., which investigated three possible combinations- oxaliplatin with 5-FU and 
leucovorin, irinotecan with 5-FU and leucovorin and irinotecan with oxaliplatin. This 
study reported a significant improvement in toxicity profile as well as a survival 
improvement with the first arm.48, 49  In summary, clinical trials of oxaliplatin combined 
with 5-FU/LV in both chemotherapy-treated and -untreated patients with colorectal 
cancer have shown the effectiveness and tolerability of the oxaliplatin/5-FU/LV 
combination.  Oxaliplatin is approved in the United States for the treatment of advanced 
colorectal cancer and for the adjuvant treatment of stage III colon cancer following 
complete resection of the primary tumor.
Oxaliplatin in cancer of the esophagus and stomach:
Oxaliplatin combinations are active in the treatment of several gastrointestinal cancers 
apart from colorectal cancer, including esophageal, gastric, biliary and pancreatic tumors.  
A variety of regimens have been studied in advanced EGC in phase II trials (in 
combination with 5-FU, capecitabine, irinotecan, docetaxel), with response rates ranging 
from 40-60%.8-13, 50  An overview of the treatment options in these diseases is provided in 
reference #4.  The efficacy of oxaliplatin in advanced EGC has been confirmed in two 
phase III trials.7, 51  In the REAL-2 trial, therapy-naïve patients were randomized to one 
of four combination treatment arms, aiming to examine the efficacy of oxaliplatin (as 
compared to cisplatin) and capecitabine (as compared to 5-FU).  There was no significant 
difference in response rates or progression-free survival between the arms.  Survival in 
the two oxaliplatin-containing arms was comparable to the cisplatin-containing arms, 
with less neutropenia, alopecia, thromboembolism and renal insufficiency, but greater 
neuropathy and diarrhea.  Overall survival for the epirubicin, oxaliplatin and capecitabine 
(EOX) arm was 11.2 months (significantly longer compared to the standard ECF arm, 9.9 
months) on secondary analysis.7 Similar results of were found in the AIO study 
comparing oxaliplatin with cisplatin, when combined with leucovorin-modulated 5-FU.51  
Oxaliplatin is listed in the NCCN Drugs and Biologic CompendiumTM for the treatment 
of advanced esophageal or gastric cancer and in the pre-operative or definitive treatment 
of localized disease (with or without radiotherapy).     
HANDLING AND STORAGE OF OXALIPLATIN
Oxaliplatin will be obtained from commercial supply for this clinical trial.
  
Concentrate for solution for infusion:
Oxaliplatin is supplied in clear, glass, single-use vials containing 50 mg or 100 mg 
oxaliplatin as a sterile, preservative-free, aqueous solution at a concentration of 5 mg/ml.  
Tartaric acid, NF, Water for Injection, USP, and Sodium Hydroxide, NF are used as 
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inactive ingredients and/or in combination as a buffering system
NDC 61703-363-18: 50 mg / 10 ml single-use vial individually packaged in a carton.
NDC 61703-363-22: 100 mg / 20 ml single-use vial individually packaged in a carton.
Preparation of Infusion Solution:
Concentrate for solution for infusion
i.
Do not freeze and protect from light the concentrated solution.
ii.
A final dilution must never be performed with a sodium chloride solution or other 
chloride-containing solutions.
iii.
The solution must be further diluted in an infusion solution of 250-500 mL of 5% 
Dextrose Injection, USP.
iv.
After dilution with 250-500 mL of 5% Dextrose Injection, USP, the shelf life is 
6 hours at room temperature [20-25°C (68-77°F)] or up to 24 hours under 
refrigeration [2-8°C (36-46°F)].
v.
After final dilution, protection from light is not required.
Incompatibilities:
Oxaliplatin is incompatible in solution with alkaline medications or media (such as basic 
solutions of 5-FU) and must not be mixed with these or administered simultaneously 
through the same infusion line. The infusion line should be flushed with D5W prior to 
administration of any concomitant medication.  Parenteral drug products should be 
inspected visually for particulate matter and discoloration prior to administration and 
discarded if present.
Needles or intravenous administration sets containing aluminum parts that may come in 
contact with oxaliplatin should not be used for the preparation or mixing of the drug. 
Aluminum has been reported to cause degradation of platinum compounds.
Handling and Disposal:
As with other potentially toxic anticancer agents, care should be exercised in the handling 
and preparation of infusion solutions prepared from oxaliplatin. The use of gloves is 
recommended. If a solution of oxaliplatin contacts the skin, wash the skin immediately 
and thoroughly with soap and water. If oxaliplatin contacts the mucous membranes, flush 
thoroughly with water.
Institutional procedures for the handling and disposal of anticancer drugs should be 
considered.
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Storage: 
Concentrate for solution for infusion:
Store under normal lighting conditions at 20°C-25°C (68°F-77°F); excursions permitted 
to 15-30°C (59-86°F). Do not freeze.
7. Study Population
I.
Stage:  Advanced esophago-gastric cancer (metastatic or unresectable)
II.
Major Inclusion / Exclusion Criteria
Inclusion
1. Histologically confirmed carcinoma of the esophagus, stomach or gastro-
esophageal junction that is metastatic, or locally advanced and inoperable for 
cure.  Histologic sub-types permitted include adenocarcinoma, squamous-cell 
carcinoma, or undifferentiated carcinoma.  Small-cell carcinoma variant is not 
eligible.
2. No previous systemic therapy for metastatic or recurrent disease.  Therapy 
(chemotherapy, radiotherapy, or both) administered in the neo-adjuvant, 
adjuvant, or definitive setting for previously localized disease is permitted, 
provided it was completed more than 6 months prior to enrollment.  Palliative 
radiotherapy is permitted provided it is completed > 3 weeks prior to study 
therapy initiation.
3. ECOG performance status 0-2.
4. Life expectancy > 12 weeks.
5. Age > 18 years.
6. Adequate hematologic parameters (hemoglobin > 9 g/dl, absolute neutrophil 
count > 1500/mm3, platelet count > 100,000/mm3).
7. Adequate biochemical parameters (serum creatinine < institutional upper 
limit normal (ULN), bilirubin < 1.5 X ULN, transaminases < 3 X ULN; for 
documented liver metastases, transaminases up to 5 X ULN is permitted).
8. No evidence of > grade 2 peripheral neuropathy.
9. Patients with reproductive potential must be willing to use an adequate 
contraceptive method (e.g., abstinence, intrauterine device, oral 
contraceptives, barrier device with spermicide or surgical sterilization) during 
treatment and for three months after completing treatment.  A negative 
pregnancy test is required for women of child-bearing potential.  Nursing 
women are ineligible.
10. Written, informed consent.
Exclusion
1. Hypersensitivity to platinum compounds.
2. Uncontrolled inter-current illness including but not limited to active infection, 
symptomatic congestive heart failure, unstable angina, uncontrolled cardiac 
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arrhythmia, or psychiatric illness that would limit compliance with study 
requirements.
3. Presence of brain metastases.
4. Patients with third-space (pleural, peritoneal) fluid not controllable with usual 
drainage methods are not eligible.
5. History of second primary malignancy within 3 years prior to enrollment, 
except for in-situ cervix carcinoma or non-melanoma skin cancer.
6.
Undergone an allogeneic stem cell transplant 
8. Treatment Plan
8.1
Premedication
Folic acid (1-1.25 mg/d) will be given orally daily starting at least 10 days prior to 
the first dose of pralatrexate and must be continued during the full course of 
treatment with pralatrexate, and for 30 days after the last dose of pralatrexate.
Please see appendix 1: Diary for folic acid medication   
Vitamin B12 (1000 mcg) will be administered intra-muscularly no more than 10 
weeks prior to the first dose of pralatrexate and repeated every 8-10 weeks 
thereafter while on treatment with pralatrexate.  Subsequent B12 injections may be 
given the same day as pralatrexate.  
8.2
Chemotherapy
Following appropriate anti-emetic therapy (to include a 5-HT3 antagonist and a 
steroid), pralatrexate will be administered intravenously in a dose of 120 mg/m2 
over 3-5 minutes followed by oxaliplatin at 85 mg/m2 intravenously over 2 hours.  
Cycles will be repeated every 2 weeks until disease progression, undue toxicity, 
or patient/physician discretion.  For patients meeting response criteria to continue 
therapy, oxaliplatin will be discontinued after 12 cycles (maximum cumulative 
dose = 1020 mg/m2) to reduce the risk of peripheral neuropathy.
The following table outlines the cohorts that may be examined based on tolerance 
of the combination.  Details are outlined in the safety lead-in section 11 of the 
study.  
Cohort 1
Cohort -1 (if 
needed)
Cohort -2 (if 
needed)*
Oxaliplatin
85mg/m2
85mg/m2
65mg/m2
Pralatrexate
120mg/m2
100mg/m2
80mg/m2
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*Examination of cohort -2 will only be explored at the discretion of the 
investigator following discussions with the biostatistician, NCCN and Allos 
Therapeutics, Inc. 
General Guidelines for Chemotherapy Use:
•
The dose of chemotherapy may be rounded off (up or down) to the nearest 
whole number or for purposes of minimizing wastage from a drug vial so 
long as the dose is within 5% of the actual calculated dose.
•
It is not necessary to change the dose of chemotherapy administered in 
successive cycles unless the calculated dose changes by > 10%.
•
There is no clearly documented adverse impact of treatment of obese 
patients when dosing is performed according to actual body weight.  
Therefore all calculations will be based solely on the patients’ actual body 
weight for the purpose of determining the body surface area.  This will 
help minimize calculation errors and possible introduction of variation in 
dose administration.  Failure to adhere to this will be considered a major 
protocol violation.
•
Patients should be counseled to avoid ice-chips, cold drinks, and exposure 
to cold water or air as the neurotoxicity seen with oxaliplatin is often 
exacerbated by cold exposure.  The duration of this cold-related 
neuropathy is not well defined and patients should exercise caution 
regarding cold exposure during the treatment cycle.
8.3
Duration of Therapy
In the absence of treatment delays due to adverse events, treatment may continue 
until one of the following criteria applies:
•
Disease progression,
•
Intercurrent illness that prevents further administration of treatment,
•
Unacceptable adverse events(s),
•
Patient decides to withdraw from the study, or
•
General or specific changes in the patient's condition render the patient 
unacceptable for further treatment in the judgment of the investigator.
8.4
Duration of Follow-Up
Patients who have completed protocol therapy will be followed for toxicity 
assessment and resolution for up to 30 days after the last dose of study drug(s).  
This may be extended appropriately in case of non-resolution of toxicity.  
Subsequent follow-up will be per standard of care in order to record survival data.  
This will be until a maximum of 5 years or death, whichever occurs first.
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9. Supportive Care Guidelines
9.1.
Patients should receive full supportive care, including transfusion of blood and 
blood products, antibiotics and anti-emetics where deemed medically appropriate.  
The use of aprepitant is permitted if clinically indicated.
9.2.
Growth Factor Use
The use of erythropoietin (EPO) and darbepoetin is permitted at the discretion of 
the treating physician.
The use of filgastrim (G-CSF), pegfilgastrim and sargramostin (GM-CSF) is 
discouraged.  They may not be used:
•
To avoid dose delays or reductions
•
Prophylactically for concern of myelosuppression
•
For the routine treatment of febrile neutropenia (except in the following 
circumstances)
However the use of CSFs is permitted per ASCO guidelines, e.g., patients with 
indicators of poor prognosis such as pneumonia, hypotension, multi-organ 
dysfunction and invasive fungal infection.  In these cases, treating physician 
discretion will prevail.  The reason and duration for CSF use should be recorded.
  
10. Required Data and Study Calendar
Guidelines for pre-study testing
•
To be completed within 14 days of registration
o
Required laboratory data, history and physical
•
To be completed within 28 days of registration
o
Any imaging used for staging purposes
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Prior to 
Registration*
Day 1 of every 
cycle*
Follow-
up**
Tests and Observations
History / Examination
Weight / BSA
Performance Status
Toxicity Assessment
Neurologic Examination
X
X
X
X
X
X
X
X
X
X
X
X
Laboratory Studies
CBC / Differential1
Comprehensive Metabolic Panel1
LDH
PT/INR
Pregnancy Test2
X
X
X
X
X
X
X
X
Staging / Imaging
Contrast CT (CAP; Neck if indicated) 
or MRI3
PET (if indicated)3
X
X
X3
Correlative Studies
EGD with biopsy4
Blood for pharmacogenomics (5ml)5
X
X
 NOTE: If treatment day (day 0 of a given cycle) falls on a designated holiday, treatment can be 
administered on days -1 to +2, provided treatment parameters have been met.. 
* Tests, observations, and laboratory studies completed within 7 days prior to the first day of treatment 
need not be repeated; one cycle is defined as 2 weeks
** Upon completion of protocol related treatment until 30 days after the last dose of study drug(s); this can 
be repeated at weekly or biweekly intervals till resolution of drug-related toxicity.  At least one visit must 
occur on or around the 30 day mark (or 7 days beyond)
1.
Can be repeated as needed during a treatment cycle if indicated (e.g. toxicity purposes)
2.
For women of child-bearing potential (urine or serum)
3.
Preferred imaging is a contrast-enhanced CT scan (chest, abdomen and pelvis; neck, if indicated).  In 
case of contrast allergy despite the use of recommended prophylaxis, a contrast- MRI or an FDG-PET 
may be used for staging and follow-up provided target lesions can be accurately measured.  Imaging to 
be obtained every 8 weeks (+/- 7 days) while on treatment, even if dose delays or interruptions in 
therapy have occurred. 
4.
For patients whose primary tumor tissue block (formalin-fixed paraffin embedded) is not readily 
available.  This can be performed at any time prior to start of therapy, but only after informed consent 
for the protocol has been obtained.  Non-availability of primary site tumor tissue will not preclude 
enrollment on this study towards the primary end-point of efficacy.  These patients will be excluded 
from the micro-RNA correlative study analysis (section 4.2.4) 
5.
Can be done at any time prior to or during protocol therapy – treatment does not affect genetic 
polymorphisms 
11. Safety Lead-In Cohort
This initial portion of the study will be limited to the first 6 patients at cohort 1 for 
purpose of toxicity evaluation of the combination of pralatrexate and oxaliplatin.  This 
dose level will be considered tolerable following completion of two cycles (28 days) of 
treatment in 5 out of 6 patients in the absence of dose limiting toxicity (DLT).  If > 2 
patients experience DLT at this dose level, then 6 additional patients will be enrolled at 
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cohort -1.  Cohort -1 will be considered tolerable if < 1 patient experiences a DLT.  If so, 
the remainder patients will be accrued at this level.  If this criterion is not met (i.e. > 2 
DLTs observed), then further examination of a lower dose (cohort -2) will be explored at 
the discretion of the investigator following discussions with the biostatistician, NCCN 
and Allos Therapeutics, Inc.
Dose-limiting toxicity in this safety lead-in cohort will be defined as any of the 
following conditions related to study treatment occurring within the first 28 days of 
treatment (2 cycles):
1.
Grade 4 neutropenia lasting > 7 days
2.
Febrile neutropenia, defined as grade 3 or 4 neutropenia associated with a fever of   
> 100.4 F ( = 38.0 C)
3.
Grade 4 thrombocytopenia
4.
Any > grade 3 non-hematologic toxicity with the following exceptions:
a. Grade 3 diarrhea, nausea or vomiting in the absence of optimal anti-diarrheal or 
anti-emetic treatment, provided toxicity resolves within 7 days and no dose reduction 
is required.
b. Grade 3 hypocalcemia, hypokalemia, hypomagnesemia, hyponatremia, or 
hypophosphatemia which responds to medical intervention, provided toxicity 
resolves within 7 days and no dose reductions are required. 
5.
Treatment delay of > 14 days for toxicity
Hypersensitivity reactions to oxaliplatin will not constitute a DLT – if this adversity 
necessitates discontinuation of the drug within the first 28 days of therapy for this cohort, 
the patient will be replaced.  Patients who experience a DLT will be dose reduced to the 
next lower dose cohort for continuation on the study.  If an observed toxicity is clearly 
known to be within the expected toxicity profile of one of the study drugs, the attribution 
of this towards a DLT would be considered appropriate only if the severity of observed 
toxicity is greater than what would be typically observed with that drug alone.
Accrual will be halted after the first 6 patients are enrolled to permit assessment of 
toxicity of the combination.  At least 28 days must have elapsed following the initiation 
of therapy for the sixth patient to enable complete evaluation.  Patients enrolled in the 
safety lead-in part will count towards full accrual, including primary and secondary 
objectives.  If a patient in this cohort does not complete 28 days of therapy, he/she will be 
replaced provided the discontinuation was not secondary to toxicity.  The data on the first 
six evaluable patients and interpretation thereof will be communicated to the 
biostatistician, RPCI Phase I committee and the IRB before continuation of enrollment 
commences.
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12. Dose Delays and Dose Modifications
A new cycle of treatment may not begin until the ANC is > 1500/mm3 and the platelet 
count is > 100,000/mm3, and any treatment-related non-hematologic toxicity (except 
sensory neuropathy – see below) has resolved to < grade 1.  In this case, weekly 
evaluations (clinical and laboratory) will be required to assess for resumption of therapy.  
Prior to any pralatrexate dose, mucositis must be < grade 1.  Assessment and grading of 
toxicity will be per NCI CTCAE Version 4.0.
Dose modifications and/or delays will be based on laboratory parameters obtained on day 
1 of a given cycle, or based on nadir counts, if obtained (or worst non-hematologic 
toxicity) during that cycle.  Blood counts obtained within 1 day prior may also be used to 
determine continuation/modification of dosing.  As an example – a patient enrolled who 
lives some distance away from RPCI may have counts done closer to home on the day 
prior to determine eligibility for treatment on a given day.
In the event of adverse events, dose modifications should apply as per this section with 
the following guidelines: 
•
For toxicities, which are considered by the investigator unlikely to develop into 
serious, or life–threatening events (e.g. alopecia, altered taste etc.), treatment 
will be continued at the same dose without reduction or interruption. In 
addition, no dose reductions or interruptions will be required for anemia as it 
can be satisfactorily managed by transfusions.  
•
For any event, which is apparent at baseline, the dose modifications will apply 
according to the corresponding shift in toxicity grade, if the investigator feels it 
is appropriate.  (e.g. if a patient has grade 1 asthenia at baseline which increases 
to grade 2 during treatment, this will be considered as a shift of 1 grade and 
treated as a grade 1 toxicity for dose modification purposes).
•
If in the opinion of the investigator, a noted toxicity is considered solely due to 
one drug, then the dose of the other drug need not be modified.
If the dose is modified to manage an adverse event, it is recommended that the patient be 
seen by the investigator prior to re-starting study drug.  Once a dose has been reduced it 
will not be increased at a later time.   If toxicity or other reason requires a dosing 
delay/interruption of both drugs of more than four weeks, the patient will be withdrawn 
from protocol therapy and the reason recorded. 
The following dose modification table will be followed (for patients with starting dose at 
cohort 1):
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Starting Dose
First Dose 
Reduction
Second Dose 
Reduction
Oxaliplatin
85mg/m2
65mg/m2
50mg/m2
Pralatrexate
120mg/m2
100mg/m2
80mg/m2
The following dose modification table will be followed (for patients with starting dose at 
cohort -1):
Starting Dose
First Dose 
Reduction
Second Dose 
Reduction
Oxaliplatin
85mg/m2
65mg/m2
50mg/m2
Pralatrexate
100mg/m2
80mg/m2
60mg/m2
The patient will be taken off protocol therapy if dose reduction beyond dose level -2 is 
required.
12.1
Dose modification for hematologic toxicity:
12.1.1 Grade 2 neutropenia or thrombocytopenia:  No change in dose for 
subsequent cycles.  If count recovery takes > 2 weeks, reduce both drug 
doses by one dose level.
12.1.2 Grade 3 neutropenia (uncomplicated):  No change in dose for 
subsequent cycles.  If count recovery takes > 2 weeks, reduce both drug 
doses by one dose level.
12.1.3 Grade 4 neutropenia, febrile neutropenia (defined as ANC < 1000 and 
fever > 38.0 C) or grade 3/4 thrombocytopenia:  Decrease by one dose 
level (both drugs)
12.1.4 Anemia, regardless of grade, will not warrant dose modification unless 
deemed necessary by the investigator.
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12.2
Dose modification for mucositis:
Mucositis Grade on Day 
of Treatment
Action1
Dose upon recovery to ≤ Grade 1
Grade 2
Omit dose
Continue prior dose
Grade 2 recurrence
Omit dose
↓ both drugs by one dose level
Grade 3
Omit dose
↓ both drugs by one dose level
Grade 4
Omit dose
↓ both drugs by two dose levels*
*If this occurs at dose level -1, discontinue all protocol therapy
1 For both drugs
Management of mucositis:  A suggested regimen is to start oral leucovorin 25mg q6 
hours X 3-5 days for > grade 2 mucositis.  This should be discontinued at least 48 hours 
prior to the next planned dose of pralatrexate.53
12.3
Dose modification for diarrhea
Diarrhea Grade on Day 
of Treatment
Action1
Dose upon recovery to ≤ Grade 1
Grade 2
Omit dose
Continue prior dose
Grade 2 recurrence
Omit dose
↓ both drugs by one dose level
Grade 3
Omit dose
↓ both drugs by one dose level
Grade 4
Omit dose
↓ both drugs by two dose levels*
*If this occurs at dose level -1, discontinue all protocol therapy
1 For both drugs
12.4
Dose modification for nausea and emesis
For grade 3 or 4 nausea and emesis that persist despite the use of aprepitant and 
steroids, reduce the dose of both drugs by one dose level.
12.5
Dose modification for hepatic toxicity
For ≥ Grade 3 elevation in AST (SGOT), ALT (SGPT), alkaline phosphatase or 
bilirubin, the dose of oxaliplatin and pralatrexate will be reduced by one level 
during the next and subsequent cycles of therapy.
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12.6
Dose modifications for other non-hematologic toxicity
Toxicity Grade on Day of 
Treatment
Action1
Dose upon recovery to 
≤ Grade 1
Grade 2
Omit dose*
Same
Grade 3
Omit dose
↓ both drugs by one dose 
level
Grade 3 re-occurrence at 
reduced dose
Omit dose
↓ both drugs by one dose 
level
Grade 4
Omit dose
↓ both drugs by two dose 
levels**
*  If clinically significant; in the event of recurrent grade 2 toxicity resulting in treatment delays, a 
     reduction of both drugs by one dose level may be unndertaken     
**If this occurs at dose level -1, discontinue all protocol therapy
1 For both drugs
12.7
Toxicities unique to oxaliplatin
12.7.1 Neurotoxicity
Evaluation of neurologic toxicity should occur prior to each oxaliplatin dose.  The 
patient should be questioned for neurologic symptoms and an appropriate 
neurologic examination performed.  See Table below for an appropriate 
neurologic toxicity grading scale. 
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Oxaliplatin Dose Modifications for Neurologic Toxicity
Duration of Toxicity
Toxicity
1 - 7 Days
> 7 Days
Persistenta
between Cycles
Paresthesias/dysesthesiasb of 
short duration that resolve and do 
not interfere with function 
(Grade 1)
no change
no change
no change
Paresthesias/dysesthesiasb 
interfering with function, but not 
activities of daily living (ADL) 
(Grade 2)
no change
no change
↓ one dose level
Paresthesias/dysesthesiasb with 
pain or with functional 
impairment that also interfere 
with ADL (Grade 3)
1st time: ↓ one dose level
2nd time: ↓one dose level
1st time: ↓one dose level
2nd time: ↓ one dose level
Stop (continue pralatrexate 
only)
Persistent 
paresthesias/dysesthesias that are 
disabling or life-threatening
(Grade 4)
Stop
(continue pralatrexate 
only)
Stop
(continue pralatrexate 
only)
Stop
(continue pralatrexate 
only)
Pharyngo-laryngeal dysesthesias
SEE BELOW
aNot resolved by the beginning of the next cycle.
bMay be cold-induced.
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Laryngopharyngeal Dysesthesia:
An unusual laryngopharyngeal dysesthesia (LPD), a loss of sensation of breathing 
without any objective evidence of respiratory distress (laryngospasm, bronchospasm or 
hypoxia) has also been observed.  This neurotoxicity may be induced or exacerbated 
upon exposure to cold and should be distinguished from a hypersensitivity reaction.  If a 
patient develops LPD, the patient’s oxygen saturation should be evaluated via a pulse 
oximeter and, if normal, reassurance, a benzodiazepine or other anxiolytic agent should 
be considered and the patient should be observed in the clinic until the episode has 
resolved.  The oxaliplatin infusion may then be continued at a reduced rate, 33% of the 
original rate.  Because this syndrome may be associated with the rapidity of oxaliplatin 
infusion, subsequent doses of oxaliplatin should be administered as 6-hour infusions.  To 
minimize the risk of LPD, patients will be instructed to avoid ice and cold drinks the day 
of treatment. 
Hypersensitivity reactions:
Oxaliplatin, as is the case with all platinum-containing compounds, is associated with a 
measurable (approximately 11%) incidence of hypersensitivity reactions, usually after 
multiple doses of treatment. This may present as bronchospasm, hypotension, and even 
hemolytic anemia.  Pretreatment with glucocorticoids and antihistamines may be useful 
for some patients, but may not always prevent the development of anaphylactoid 
reactions, especially in patients with a prior history of hypersensitivity to this agent.  For 
patients who have experienced a Grade 1 or 2 acute hypersensitivity reaction that is 
assessed as related to oxaliplatin administration, the following premedication is 
recommended prior to each subsequent dose of oxaliplatin (patients who have grade 3 or 
4 acute hypersensitivity reactions should discontinue oxaliplatin therapy):  
Dexamethasone 20 mg PO or IV, 12 and 6 hours prior to the oxaliplatin dose;
OR
Dexamethasone 20 mg PO or IV, as well as diphenhydramine 50 mg IV, and one of the 
following: cimetidine 300 mg IV, ranitidine 50 mg IV, or famotidine 20 mg IV 30-60 
minutes prior to oxaliplatin administration. If these prophylactic measures fail to prevent 
oxaliplatin-related hypersensitivity, therapy with oxaliplatin should be discontinued.  In 
this case, pralatrexate alone will be continued.
Pulmonary Fibrosis:
In the case of unexplained respiratory symptoms such as nonproductive cough, dyspnea 
or radiological pulmonary infiltrates, oxaliplatin should be discontinued until further 
investigation excludes interstitial pulmonary fibrosis.  If interstitial pulmonary fibrosis is 
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Hemolytic-Uremic Syndrome:
Upon monitoring weekly hematologic and renal parameters, if new onset of renal failure 
and/or hemolytic anemia is found, oxaliplatin will be held until the etiology of the renal 
failure and/or hemolytic anemia can be determined.  Laboratory parameters that may be 
be used to determine whether hemolytic uremic syndrome (HUS) is present include: 
CBC, differential, platelets, PT, PTT, fibrinogen, FDP, ATIII, VWF, ANA, RhF, C3, C4, 
CH50, antiplatelet antibodies, platelet-associated IgG, and ciruculating immune 
complexes.  If oxaliplatin is determined to be the cause of renal failure, hemolytic anemia 
or HUS, the drug will be permanently discontinued.
13. Endpoints
Primary: The primary end-point of this phase II trial is the overall response rate (ORR) 
to combination pralatrexate and oxaliplatin in advanced EGC.  This will be determined 
using the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 
guidelines every 8 weeks while on treatment.52  Objective responses will be confirmed 4 
weeks after first documentation of response.
Secondary: The toxicity of this regimen will be recorded for all patients who received at 
least one dose of therapy.  Other end-points will include calculation of time-to-
progression and overall survival.  Definitions of these variables are in the statistical 
section.
Correlative: The pharmacogenomic and tissue micro-RNA profiling end-points of this 
trial are outlined in the respective correlative science section.
14. Statistical Analysis
The study is a two-stage, single arm, unblinded Phase II trial allowing for early 
termination in case of futility.  In the standard of care therapy, overall response rates 
(complete response + partial response) for advanced esophago-gastric cancer are around 
0.20.  An exact two-stage design proposed by Kepner and Chang tests the null hypothesis 
that the proposed treatment has an overall response rate that is at-most equal to the 
standard of care, against an alternative that it is greater.54  The following study design has 
80% power to detect a 0.40 overall response rate with the study treatment, while 
controlling to 5% the probability of erroneously finding a truly ineffective treatment as 
worthy of further research.
Seventeen eligible patients will be enrolled in the first stage of the trial.  If a maximum of 
3 of these patients achieve complete or partial response, the treatment will be declared 
ineffective, and the study terminated.  If 4 or more patients achieve complete or partial 
response, an additional 16 patients will be enrolled.  If 11 or more of the 34 total patients 
achieve complete or partial response, the treatment will be deemed effective and 
considered for further study.
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Time to tumor progression (TTP) will be measured from the date of study enrollment to 
the first observation of progressive disease.  Overall survival (OS) will be measured from 
the date of study enrollment to the time of death from any cause.  These variables will be 
estimated using the Kaplan-Meier method.  Statistics describing the time to event 
distributions will be obtained from Kaplan-Meier methods and Proportional Hazards 
models.  Continuous variables will be summarized with commonly used statistics (mean, 
standard deviation, median, etc.), with sub-group associations tested using the Wilcoxon 
Rank Sum test.  Categorical variables will be summarized in contingency tables, with 
associations of interest assessed using Fisher’s Exact Test.  Given the exploratory nature 
of this study, all p-values less than 0.05 will be deemed statistically significant.  No 
adjustment will be made to the significance threshold to control for the effects of multiple 
testing on the overall Type I error rate.
The analyses for the correlative science studies are outlined in the correlative study 
section.
15.
Correlative Studies
15.1
Background:
Pralatrexate like pemetrexed is a novel antifolate with activity in non small cell lung 
cancer (NSCLC), Hodgkin’s disease, mesothelioma and bladder cancer. Pralatrexate is an 
analog of methotrexate and acts as a potent dihydrofolate reductase (DHFR) inhibitor. 
Studies indicate that pralatrexate selectively enters cells expressing reduced folate carrier 
(RFC-1) which is encoded by SLC19A1; and its increased retention in cells results from 
its high affinity for folylpolyglutamate synthase (FPGS). Like pemetrexed, the steady 
state accumulation of pralatrexate polyglutamates will depend primarily on 
activation/inactivation processes by FPGS and GGH respectively.55-57  In previous studies 
with pemetrexed in NSCLC we identified correlations between haplotype-tagged single 
nucleotide polymorphisms (ht-SNPs) in SLC19A1, the gamma glutamyl hydrolase 
gene,GGH, and FPGS and efficacy as well as toxicity.  In this proposal, we hypothesize 
that functionally relevant polymorphisms in these 3 genes as well as selected additional 
tagSNPs for genes from the folate pathway such as MTHFR and TYMS and DHFR 
either singly or in combination, play a role in the efficacy and/or toxicity of 
pralatrexate (Correlative Section Specific Aim 1).
MicroRNAs (miRNAs) are small non-coding RNAs that were first described in the 
nematode C. elegans more than a decade ago58, and are now known to regulate the 
expression of protein coding genes in various organisms. Over 800 miRNAs have been 
identified in humans. While changes in expression patterns of mRNA have been shown to 
be promising in chemosensitivity prediction in several human cancers, there are 
significant problems in their utilization in everyday clinical use. The requirement of fresh 
frozen RNA that is carefully preserved is one such significant limitation. MicroRNAs are 
stable to degradation over long periods of time.59  This enables the use of RNA extracted 
from paraffin-embedded tissue, thereby enhancing and simplifying clinical use.  The 
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number of miRNA known at this time is smaller, making miRNA profiles more robustly 
analyzable statistically than mRNA profiles. Therefore a microRNA signature is 
potentially more clinically feasible for everyday use. Limited data exists for the 
utilization of microRNA signatures for chemosensitivity prediction. As shown in the 
figure below, cell lines with increasing resistance (increasing IG50) show different 
microRNA profiles. This difference may be utilized in chemosensitivity prediction.  In 
the Correlative Section Specific Aim 2, we hypothesize that response to pralatrexate 
can be predicted by microRNA expression profiling of the epithelial component of the 
tumor.
 
15.2
Preliminary Data:  
We have resequenced FPGS and obtained polymorphism (SNP) information.60  We also 
utilized data from NCBI and HapMap databases to obtain polymorphism information on 
GGH and SLC19A1.  We identified SNPs in these three candidate genes, SLC19A1 and 
GGH and FPGS, and genotyped for the SNPs in two studies with pemetrexed–treated 
patients.61-63  In these small studies,SNPs in SLC19A1, FPGS and GGH correlated with 
efficacy and toxicity of pemetrexed. In the pemetrexed-gemcitabine study, patients 
heterozygous for polymorphisms in FPGS and GGH had more grades 3/4 adverse events 
including SGPT (ALT) elevation, dyspnea and fatique, although not statistically 
significant. Similarly, polymorphisms in GGH associated with confirmed response.62  As 
well, three SNPs in SLC19A1 predicted for statistically significant differences in overall 
survival in the study: IVS4(2117)T>C; IVS5(9148) A>C and Exon6(2522) C>T, p=0.03.  
A similar analysis in our recently completed phase II study (50 patients) of pemetrexed in 
combination with bevacizumab in second-line NSCLC also identified SNPs in FPGS, 
GGH and SLC19A1 which correlated with toxicity and efficacy.63 
15.3
Study Design and Methods (Pharmacogenomics):
Specific Aim 1:  Genotype DNA samples from patients enrolled in the clinical trial 
for tagged SNPs in candidate genes responsible for the activation, inactivation and 
transport of pralatrexate. The genes are FPGS, GGH and SLC19A1.  We propose to 
evaluate the possible contribution of pharmacogenetics (PG) to variable molecular 
responses and toxicity of pralatrexate by correlating clinical activity and toxicity with 
intragene haplotypes in FPGS, GGH and SLC19A1. The tagged SNPs will be correlated 
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with toxicity (myelosuppression, mucositis, LFT elevation) and efficacy (objective 
responses, TTP) of pralatrexate.   
15.3.1 FPGS, SLC19A1, GGH and other folate pathway polymorphisms: We have identified 
tagSNPs from our resequencing data and the HapMap mining studies for FPGS, 
SLC19A1 and GGH.60, 62, 63  Samples from this clinical study will be genotyped for all 
tagSNPs identified. Other polymorphisms that will be genotyped for include well-known 
SNPs in MTHFR and TYMS, namely C677T, A1298C in MTHFR and the 28bp-repeat 
polymorphism, and 1494del6 of the TYMS, as well as SNPs in DHFR. These SNPs have 
been associated to varying extents with methotrexate toxicity and efficacy.64-68  A list of 
49 tagSNPs to be genotyped is show below. To generate these tagSNPs, the SNP 
databases including HapMap, dbSNP and SeattleSNP were used. The SNP genotype 
datasets for the genes above were loaded into Haploview v 4.069 and polymorphisms with 
frequencies greater than 5% were selected for haplotype analysis at an r2 threshold of 0.8.  
Haplotypes close to or above 2% were then organized into single blocks from which ht-
SNPs were derived. In addition, the tagger software was used to generate additional 
tagSNPs.
Genes showing TagSNPs to be genotyped
Gene 
FPGS
GGH
SLC19A1
MTHFR
TYMS
DHFR
dbSNP id
rs# /
or SNP location
5FR -573
rs11545077 
rs914232
rs2184226
rs2853533
rs2560424
rs10760502
rs3780126
rs2838958
rs1537514
rs502396
rs1677693
IVS1 (28)
rs11990678 
rs2297291
rs4846049
rs2244500
rs11951910
IVS9 (48)
rs3780130
rs2838956
rs2274976
rs2847153
rs11742668
rs7010484 
rs3788189
rs3818762
rs2847149
rs13161245
rs11995525
rs1051298
rs1476413
rs15872
rs10072026
rs12677953 
rs1801131
rs699517
rs1650697
rs12121543
rs2853542
rs6541003
rs34489327
rs1801133
rs17421511
rs11121832
rs7533315
rs9651118
rs17367504
rs13306561
15.3.2 Sample Collection and genotyping:  Five (5) ml of blood will be collected into an 
EDTA tube at baseline or at any time during the study (since therapy does not affect gene 
polymorphisms). Genomic DNA will be extracted and stored till analysis as previously 
described.63 All genotyping are PCR-based and will be performed, using established 
methods at RPCI on the Sequenom MassARRAY platform as described in Adjei et al., 
2009.63 Network Sites please see Appendix 2 for shipping instructions.
For patients enrolled at network site(s), 5 ml of blood collected in an EDTA tube (non-
glass) should be frozen at -80°C and shipped on dry ice to the following address:
Attn: Dr. Huayi Huang
Adjei laboratory, 
GCDC Building, Room 127
Roswell Park Cancer Institute
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Buffalo, NY 14263
Phone (716) 845-7656
e-mail: huayi.huang@roswellpark.org
The site(s) will be requested to contact the receiving laboratory personnel prior to 
shipping.  It would be preferable to have samples batched for shipping.
   
15.3.3 Statistical Analysis: The primary hypothesis tested will be the association of 
dichotomous genomic events (i.e., homozygote/heterozygote, or presense/absense of 
polymorphisms, etc.) with adverse avents. If 5 or fewer subjects are observed for a 
particular genotype, e.g. homozygous, we will test for the rate of adverse events among 
heterogygotes, by a clopper pearson exact binomial test, against a 0.15 null rate of 
adverse events .  If at least 6 subjects are observed as homozygous, tests of independence 
will be performed by Fishers Exact test. Type I error control will be performed, by 
bounding the probability of more than 5 false positives to 5%. Correlations of the 
haplotype-tagged SNPs with response, OS, and TTP will be explored by multivariate 
logistic and Cox proportional hazards models,  to explore the impact of these germline 
SNPs on binary and time to event outcomes. These exploratory comparisons will be 
carried out on the entire cohort, as well as within different patient subgroups such as 
responders, and progression-free and stable patients if feasible.  
15.3.4 Sample Size: A limitation of the present study is the small sample size (34 patients), 
which lacks the power to make robust associations between SNPs and treatment outcome 
(toxicity and efficacy). We see these correlative studies as hypothesis-generating. SNPs 
identified in this study as potentially predictive of efficacy or toxicity will be tested in 
future studies.  If as many as 5 subjects are observed as homozygous, the design provides 
for 75% power to detect a true adverse event rate of 37%.  If 6 subjects are observed as 
homozygous, there will be 75% power to detect an OR of 16.5, and for 10 observed 
homozygotes, an OR of 9.44.
15.4
Study Design and Methods (MicroRNA profiling)
Specific Aim 2:  MicroRNA expression profiles generated using cell line data can 
predict response to pralatrexate chemotherapy using expression profiling of the 
epithelial component of the tumor isolated using laser-capture microdissection 
(LCM).  For these experiments, it is crucial that we use only the epithelial component of 
tumors because the cell line signatures that we will attempt to validate are based on only 
the epithelial component. There is data that strongly suggests that the gene expression of 
whole tumors is different from that of the epithelial component isolated by LCM70.  This 
is especially important when the gene expression measurements are those of miRNA 
because the changes seen in miRNA expression classifiers typically have small fold 
changes and using whole tumors may therefore mask relevant signals. Network Sites 
please see Appendix 2 for shipping instructions.
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15.4.1 Experimental plan:  Existing chemosensitivity data of the NCI-60 cell line panel to 
pemetrexed will be used to generate a miRNA signature. The feasibility of using this has 
been demonstrated using publicly available chemosensitivity data 
(http://dtp.nci.nih.gov/index.html) along with publicly accessible NCI-60 miRNA 
microarray data71 in the figure above. Such an analysis will be performed using data from 
an updated miRNA microarray platform (Affymetrix Inc.) using RNA from the NCI-60 
cell line panel. This database is currently being generated by the Yendamuri laboratory 
and is scheduled to be completed by April 2010. The use of a new platform is important 
as the publicly available data was generated using older platforms using a large amount 
of input total RNA. Therefore, the older platforms cannot be used to profile RNA 
obtained by LCM.  We have tested the feasibility of generating profiles from 100 ng of 
total RNA input using the updated platform and have successfully profiled RNA from 
both the epithelial and stromal components of formalin fixed paraffin embedded (FFPE) 
lung cancer tissue. Therefore the methods required to perform the experiment have been 
standardized. Once the pemetrexed sensitivity signature is finalized based on the above 
data, the RNA obtained from the LCM-derived epithelial component of the primary 
esophago-gastric tumor tissue (FFPE) will be used to generate epithelium-specific 
miRNA profiles of the pre-treatment biopsy specimens using the Affymetrix microRNA 
microarray platform.  The primary tumor tissue will be obtained by biopsy (those with 
primary in place) or from the original FFPE block (for those patients who underwent 
prior resection, or definitive therapy without local relapse).  Non-availability of primary 
site tumor tissue will not preclude enrollment on this study towards the primary end-point 
of efficacy.  These patients will be excluded from the micro-RNA correlative study 
analysis.  The specimens will then be classified into “sensitive” and “specific” categories. 
This classification will then be compared to the clinical responsiveness of the tumor as 
judged by imaging data. We use pemetrexed sensitivity data in this proposal because 
pralatrexate chemosensitivty data of the NCI-60 cell line panel is not available and 
because of the close approximation of the mechanism of action of both drugs.
For network site(s) as well as tissue samples collected at outside institutions, the tissue 
sample (formalin-fixed paraffin block or 5-10 unstained formalin-fixed paraffin sections 
on appropriate slides along with one hematoxylin and eosin stained section) will be sent 
to the following address:
Yendamuri Laboratory
Attn:  Eric Kannisto
Roswell Park Cancer Institute
MRC 207
Elm and Carlton Streets
Buffalo, NY 14263
Phone:  (716) 845-8364
e-mail:  eric.kannisto@roswellpark.org
The site(s) will be requested to contact the receiving laboratory personnel prior to 
shipping.
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15.4.2 Statistical analysis:  The investigators realize that the limited number of samples assayed 
in this experiments permits only exploratory analyses to be performed. Initial studies will 
be conducted to test and optimize individual miRNAs for maximum sensitivity and 
specificity to predict chemosensitivity, inferring differences in the distributions of 
biomarkers with stringent statistical multiple testing error control procedures.  Affymetrix 
CEL files will be loaded into the affy bioconductor package, and preprocessed with a custom cdf 
library, using the RMA routine.  We will perform a 2-sample two-ways test of mean differences 
in mir log expression by discrete treatment outcome (toxicity or efficacy), with a wilcoxon 
rank sum test.  We will have 70% power to detect a 2 standard deviation difference in 
means, assuming 10 responders and 23 non-responders, bounding the probability of more 
than 10 false positives to 5%.  We will visually explore the distributions of candidate mir 
signature profiles, by outcome, for artifacts, outliers, symmetry and spread.  Data 
transformations,and discretizations will be evaluated for supervised and partially 
unsupervised prediction performance. We will investigate signature refinement with 
advanced penalized version of well known prediction methods, such as Bayesian 
Additive Regression Trees, Support Vector Machines, and Fisher's Discriminant with 
nested class Partitioning Around Mediods, considering univariate and multivariate 
variable selection methods.  We will evaluate the prediction performance with ROC and 
Threshold/Recall Analysis conducted under k-fold monte carlo cross validation.  
Significant differences in sensitivity, specificity and area under the curve will be inferred 
with bootstrap techniques.  Further exploration will include measuring the marginal 
predictive utility of the miRNA signatures, accounting for known and potential risk 
factors.
16. Adverse Event Reporting
IDENTIFICATION, RECORDING AND HANDLING ADVERSE EVENTS
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  An 
adverse event is any adverse change (developing or worsening) from the patient’s 
baseline (pre-treatment) condition, including intercurrent illness, which occurs during the 
course of a clinical study after treatment has started, whether considered related to 
treatment or not. All adverse events encountered during the clinical study will be reported 
on the case report form (CRF).The intensity of clinical adverse events will be graded 
according to the revised NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0, which have recommended grading. 
Adverse events not listed on the CTC grading system will be graded according to the 
following scale:
Mild:
Discomfort noticed but no disruption of normal daily activity
Moderate:
Discomfort sufficient to reduce or affect normal daily activity
Severe:
Incapacitating with inability to work or perform normal daily activity
Life–threatening:
Self–explanatory
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Adverse Events Monitoring
The collection of AE information should begin at the time the patient receives the initial 
study drug infusion and must continue throughout the study until 30 days after the last 
dose, unless specified otherwise in the protocol.  This includes pre-existing conditions or 
symptoms that worsened during the study, or whose relationship to the investigational 
product changed; but does not include pre-planned hospitalizations or procedures for pre-
existing conditions.  In addition, the investigator should notify National Comprehensive 
Cancer Network (NCCN) of any SAE or outcome that may occur after this time period 
that he/she believes to be related to pralatrexate and oxaliplatin.  All SAEs or AEs 
related to study drugs must be followed to resolution, stabilization or return to baseline, 
or deemed irreversible.  An AE report must contain the following four basic elements:  
(1) an identifiable patient; (2) a suspect medicinal product; (3) an identifiable reporting 
source; and (4) an identifiable event or outcome. All identified AEs must be recorded and 
described on the appropriate page of the CRF.  If known, the diagnosis of the underlying 
illness or disorder should be recorded in addition to the presenting symptoms.  The 
following information should be captured for all AEs:  the date of the onset and 
resolution; the severity of the event (see definitions in National Cancer Institute Common 
Terminology Criteria for Adverse Events [NCI-CTCAE] Version 4.0); the investigator’s 
opinion of the relationship of the event to the investigational product (certainly/definitely, 
probably, possibly, unlikely, or not related); the treatment required for the AE; 
information regarding resolution/outcome; and if the AE is serious, a clear identification 
of the seriousness outcome.   In addition, all Roswell Park Cancer Institute initiated trials 
will be monitored periodically by the Compliance Monitor.
Definitions 
An Adverse Event is any untoward medical occurrence in a patient administered a 
pharmaceutical product and which does not necessarily have to have a causal relationship 
with the treatment.   
Serious Adverse Events (SAE)
A serious adverse event as defined by ICH is any adverse experience that at any dose 
meets any of the following conditions:
•
results in death
•
is life-threatening (The patient was at risk of death at the time of the event. It does not  
refer to an event which hypothetically might have caused death if it were more severe)
•
requires inpatient hospitalization or prolongation of existing hospitalization
•
results in persistent or significant disability/incapacity, or
•
is a congenital anomaly/birth defect
Note: Medical and scientific judgment should be exercised in deciding whether expedited 
reporting is appropriate in other situations; for example, important medical events may 
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not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the patient or may require intervention to prevent one of the outcomes listed in 
the definition above. Any adverse event is considered a serious adverse event if it is 
associated with clinical signs or symptoms judged by the investigator to have a 
significant clinical impact.
Hospitalizations that do not meet these criteria are:
•
reasons described in the protocol, e.g., drug administration, protocol-required testing
•
social reason in the absence of an AE
•
surgery or procedure planned prior to entry into the trial
Nonserious Adverse Events
Any adverse event that is not an SAE is, by default, a non-serious AE.
An Unexpected Adverse Event is an event that is not listed in the current Clinical 
Investigator’s Brochure (CIB) / Package Insert or an event that may be mentioned in the 
CIB / Package Insert, but differs from the event because of greater severity or specificity.
For SAEs occurring in the current study, the Expectedness of an Adverse Event will 
be determined by identifying the term in the table of Expected Adverse Events by Body 
System, provided in the latest version of the CIB.  If the term is not contained in the list 
under the relevant body system, the term is to be considered UNEXPECTED. 
For comparative drugs, expectedness is determined by using the pertinent reference text: 
the US Package Insert (if marketed drug) or Clinical Investigator Brochure (if 
Investigational New Drug).
Causality is a determination of whether there is a reasonable possibility that the drug 
may have caused or contributed to an adverse event. It includes assessing temporal 
relationships, dechallenge/rechallenge information, association (or lack of association) 
with underlying diseases, and the presence (or absence) or a lack of one or more likely 
causes. The Investigator must determine if an adverse event is in some way related to the 
use of the study drug.  This relationship should be described as follows: 
Unlikely or Unrelated:  The event is clearly due to causes distinct from the use of the 
study drug, such as a documented pre-existing condition, the effect of a concomitant 
medication, a new condition which, based on the pathophysiology of the condition, and 
the pharmacology of the study drug, would be unlikely related to the use of the study 
drug.
Possible: The event follows a reasonable temporal sequence from administration of the 
study drug or the event follows a known response pattern to the study drug BUT the event 
could have been produced by an intercurrent medical condition which, based on the 
pathophysiology of the condition, and the pharmacology of the study drug, would be 
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unlikely related to the use of the study drug or the event could be the effect of a 
concomitant medication.
Probable: The event follows a reasonable temporal sequence from administration of the 
study drug and the event follows a known response pattern to the study drug AND the 
event cannot have been reasonably explained by an intercurrent medical condition or the 
event cannot be the effect of a concomitant medication
Definite: The event follows a reasonable temporal sequence from administration of the 
study drug, the event follows a known response pattern to the study drug and based on 
the known pharmacology of the study drug, the event is clearly related to the effect of the 
study drug
Unknown: Based on the evidence available, causality cannot be ascribed
Adverse Events Emerging Subsequent to Study Cessation
For 30 days subsequent to study completion or withdrawal, new onset adverse events will 
be captured.  Follow up of these events will follow the same procedure as described 
above for AEs observed during the study period.
-------------------------------------------
Safety Reporting Requirements 
-------------------------------------------
Safety Reporting Requirements and Timelines
The Investigator will utilize the MedWatch Form for the reporting of adverse events and 
follow up information to those events. 
All serious adverse events regardless of severity or relationship must be reported to Allos 
within 24 hours of the investigational staff’s knowledge.  
Spectrum Pharmaceuticals
157 Technology Drive
Irvine, CA 92618
Phone: (949) 788-6700
Fax: (949) 788-6706; be sure to retain successful fax acknowledgement
Email:  drugsafety@sppirx.com
 
And
NCCN via fax at 215-358-7699 or e-mailed to ORPReports@nccn.org
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In addition, the Investigator will adhere to the safety reporting requirements and timelines 
described in the Clinical Trial Agreement with National Comprehensive Cancer Network 
(NCCN). 
The Investigator will provide full and timely cooperation with any requests from Allos, 
governing IRB, institution, or regulatory agency with any requests regarding reports of 
individual reports of adverse events.
Network Sites please see Appendix 2 for reporting information.
-----------------------------------------------------------
Additional Requirements for IND HOLDERS  
-----------------------------------------------------------
Expedited IND Safety Reports:
Adverse events meeting certain criteria will require expedited reports to the FDA, IRB, 
and Allos Therapeutics. A case report must be one that is serious AND unexpected AND 
drug-related (to any degree) to be subject to regulations for expedited safety reports.  
Any adverse event that meets criteria as an SAE (see section entitled "Types of Adverse 
Events") is SERIOUS.
Any adverse event not already described in the study Investigator Brochure is 
UNEXPECTED.  If no formal Investigator Brochure exists, then the current, FDA-
approved product label (package insert) will be used to determine expectedness of an 
event.
 Causality assessment is described in the previous section entitled "Definition and Types 
of Adverse Events."  
Types of Expedited Safety Reports
•
7-Calendar-Day FDA Telephone or Fax Report:  
The Investigator will directly notify the FDA within 7 calendar days of any adverse event 
that is ALL of the following:
-SERIOUS due to DEATH or FATALITY, or is immediately LIFE THREATENING
-UNEXPECTED
-CAUSALLY RELATED
Notification to the FDA will be made directly to the new drug review division in the 
Center for Drug Evaluation and Research or in the product review division for the Center 
for Biologics Evaluation and Research, whichever was responsible for the review of the 
IND.
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•
15-Calendar-Day FDA Written Report:  
The Investigator will directly notify the FDA within 15 calendar days of any adverse 
event that is ALL of the following:
-SERIOUS due to NON-fatal or NON-life threatening criteria
-UNEXPECTED
-CAUSALLY RELATED
Pregnancy Statement:
During the course of the trial, all patients of childbearing potential should be instructed to 
contact the treating physician immediately if they suspect they might have conceived a 
child.  In addition, a missed or late menstrual period should be reported to the treating 
physician.  If a female patient, or an investigator, suspects that the female patient may be 
pregnant prior to administration of study drugs, the study drugs must be withheld until 
the results of a pregnancy test are available. If pregnancy is confirmed the patient must 
not receive study medications and must be withdrawn from the study.
Throughout the entire pregnancy, additional contact should be made with the patient, and 
in some cases with the healthcare provider, to identify spontaneous abortions and elective 
terminations, as well as any medical reasons for elective termination. In addition, the 
study investigator should include perinatal and neonatal outcome. Infants should be 
followed for a minimum of 8 weeks.
 If a male patient is suspected of having fathered a child while on study drugs, the 
pregnant female partner must be notified and counseled regarding the risk to the fetus. In 
addition, the treating physician must follow the course of the pregnancy, including 
prenatal and neonatal outcome. Infants should be followed for a minimum of eight 
weeks.
Upon live-birth delivery, minimum information that should be collected includes date of 
birth, length of pregnancy, sex of infant, major and minor anomalies identified at birth. 
Outcomes can be obtained via mailed questionnaires, maternal interviews, medical record 
abstraction, or a combination of these methods. All serious adverse event reports relating 
to the pregnancy, including spontaneous abortion, elective abortion and congenital 
anomalies, should be forwarded to the FDA and the NCCN and a copy sent to Allos 
Therapeutics.  
17. Criteria for Response and Progression
The primary end-point of this phase II trial is the overall response rate (ORR) to 
combination pralatrexate and oxaliplatin in advanced EGC.  This will be determined 
using the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 
guidelines every 8 weeks while on treatment.52  Objective responses will be confirmed 4 
weeks after first documentation of response.
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17.1
Definitions:
RECIST Version 1.1 differs from the original version with respect to the following:
1. Number of lesions to be assessed decreased to a maximum of 5 (from 10) with a 
maximum of 2 per organ.
2. Assessment of pathological lymph nodes is now incorporated.  Nodes that 
measure > 15mm in short axis are considered pathologic and measurable.  This 
should be included in the calculation of tumor response, if applicable.
3. Greater stringency to the definition of progressive disease which requires and 
increase by 20% in sum of the target lesions AND a 5mm absolute increase.
Changes in only the largest diameter (unidimensional measurement) of the tumor 
lesions are used in the RECIST criteria. Note:  Lesions are either measurable or non-
measurable using the criteria provided below.  The term “evaluable” in reference to 
measurability will not be used because it does not provide additional meaning or 
accuracy.
Measurable disease
Measurable lesions are defined as those that can be accurately measured in at least 
one dimension (longest diameter to be recorded) as >10 mm with CT scan (CT scan 
slice thickness no greater than 5mm) or as >10 mm by clinical examination or > 
20mm by chest X-ray.  In addition, pathologic lymph nodes measuring > 15mm in 
short axis are also considered measurable.  All tumor measurements must be recorded 
in millimeters (or decimal fractions of centimeters).
Non-measurable disease
All other lesions (or sites of disease), including small lesions (longest diameter <10 
mm using CT scan; LNs > 10mm to < 15mm), are considered non-measurable 
disease.  Blastic bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonis, inflammatory breast disease, and abdominal 
masses (identified by physical examination but not measurable by reproducible 
imaging techniques) are all non-measurable.  For lytic bone lesions or mixed lytic 
plus blastic bone lesions that have an identifiable soft tissue component reproducibly 
measurable by standard criteria, definitions of measurability above will apply.  For 
cystic lesions thought to represent malignant disease, similar criteria will apply, 
though it is preferable to use non-cystic lesions, if present, for the purpose of 
assessment and response.   
Target lesions
All measurable lesions up to a maximum of two lesions per organ and five lesions in 
total, representative of all involved organs, should be identified as target lesions and 
recorded and measured at baseline.  Target lesions should be selected on the basis of 
their size (lesions with the longest diameter) and their suitability for accurate repeated 
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measurements (either by imaging techniques or clinically).  A sum of the longest 
diameter (LD) for all target lesions will be calculated and reported as the baseline 
sum LD.  The baseline sum LD will be used as reference by which to characterize the 
objective tumor response.
Non-target lesions
All other lesions (or sites of disease) should be identified as non-target lesions and 
should also be recorded at baseline.  Non-target lesions include measurable lesions 
that exceed the maximum numbers per organ or total of all involved organs as well as 
non-measurable lesions.  Measurements of these lesions are not required but the 
presence or absence of each should be noted throughout follow-up. 
Guidelines for Evaluation of Measurable Disease
All measurements should be taken and recorded in metric notation using a ruler or 
calipers.  All baseline evaluations should be performed as closely as possible to the 
beginning of treatment and never more than 4 weeks before the beginning of the 
treatment.
Note: Tumor lesions that are situated in a previously irradiated area might or might 
not be considered measurable.  If the investigator thinks it appropriate to include 
them, the conditions under which such lesions should be considered must be defined 
in the protocol. 
The same method of assessment and the same technique should be used to 
characterize each identified and reported lesion at baseline and during follow-up. 
Imaging-based evaluation is preferred to evaluation by clinical examination when 
both methods have been used to assess the antitumor effect of a treatment.
Clinical lesions.  Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes).  In the case of skin lesions, 
documentation by color photography, including a ruler to estimate the size of the 
lesion, is recommended.
 
Chest x-ray.  Lesions on chest x-ray are acceptable as measurable lesions when they 
are clearly defined and surrounded by aerated lung.  However, CT is preferable. 
Conventional CT and MRI.  These techniques should be performed with cuts of 10 
mm or less in slice thickness contiguously.  Spiral CT should be performed using a 5 
mm contiguous reconstruction algorithm.  This applies to tumors of the chest, 
abdomen, and pelvis.  Head and neck tumors and those of extremities usually require 
specific protocols.
Ultrasound (US).  When the primary endpoint of the study is objective response 
evaluation, US should not be used to measure tumor lesions.  It is, however, a 
possible alternative to clinical measurements of superficial palpable lymph nodes, 
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subcutaneous lesions, and thyroid nodules.  US might also be useful to confirm the 
complete disappearance of superficial lesions usually assessed by clinical 
examination.
Endoscopy, Laparoscopy.  The utilization of these techniques for objective tumor 
evaluation has not yet been fully and widely validated.  Their uses in this specific 
context require sophisticated equipment and a high level of expertise that may only be 
available in some centers.  Therefore, the utilization of such techniques for objective 
tumor response should be restricted to validation purposes in reference centers. 
However, such techniques can be useful to confirm complete pathological response 
when biopsies are obtained.
Tumor markers.  Tumor markers alone cannot be used to assess response.  If 
markers are initially above the upper normal limit, they must normalize for a patient 
to be considered in complete clinical response. 
Cytology, Histology.  These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in 
tumor types, such as germ cell tumors, where known residual benign tumors can 
remain).
The cytological confirmation of the neoplastic origin of any effusion that appears or 
worsens during treatment when the measurable tumor has met criteria for response 
or stable disease can be considered to differentiate between response or stable 
disease (an effusion may be a side effect of the treatment) and progressive disease.
Response Criteria
Evaluation of target lesions
Complete Response (CR):       Disappearance of all target lesions; any pathological 
lymph node (whether target or not) must have reduction in short axis to < 10mm
Partial Response (PR):
       At least a 30% decrease in the sum of the longest 
diameter (LD) of target lesions, taking as reference the baseline sum LD
Progressive Disease (PD):
At least a 20% increase in the sum of the LD of target 
lesions, taking as reference the smallest sum LD recorded since the treatment started 
or the appearance of one or more new lesions.  In addition, the sum must also 
demonstrate an absolute increase of at least 5mm.
Stable Disease (SD):
Neither sufficient shrinkage to qualify for PR nor 
sufficient increase to qualify for PD, taking as reference the smallest sum LD since 
the treatment started
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Evaluation of non-target lesions
Complete Response (CR):
Disappearance of all non-target lesions and 
normalization of tumor marker level
Incomplete Response/Stable Disease (SD):
Persistence of one or more non-
target lesion(s) and/or maintenance of tumor marker level above the normal limits
Progressive Disease (PD):
Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions
Although a clear progression of “non-target” lesions only is exceptional, in such 
circumstances the opinion of the treating physician should prevail, and the progression 
status should be confirmed at a later time by the review panel.
Note:  If tumor markers are initially above the upper normal limit, they must normalize 
for a patient to be considered in complete clinical response.
Evaluation of best overall response
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best response 
assignment will depend on the achievement of both measurement and confirmation 
criteria.
Target 
Lesions
Non-Target 
Lesions
New 
Lesions
Overall 
Response
CR
CR
No
CR
CR
Incomplete 
response/S
D
No
PR
PR
Non-PD
No
PR
SD
Non-PD
No
SD
PD
Any
Yes or No
PD
Any
PD
Yes or No
PD
Any
Any
Yes
PD


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Notes:
•
Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be classified as 
having “symptomatic deterioration.”  Every effort should be made to document the 
objective progression, even after discontinuation of treatment.
•
In some circumstances, it may be difficult to distinguish residual disease from normal 
tissue.  When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesion be investigated (fine needle aspirate/biopsy) before 
confirming the complete response status.
Confirmatory Measurement/Duration of Response
Confirmation
This is required in the current trial as the primary end-point is response rate.  To be 
assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat assessments that should be performed 4 to 6 weeks after the criteria for response 
are first met.  In the case of SD, follow-up measurements must have met the SD criteria 
at least once after study entry at a minimum interval of 6 to 8 weeks.
Duration of overall response
The duration of overall response is measured from the time measurement criteria are met 
for CR or PR (whichever is first recorded) until the first date that recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).
The duration of overall CR is measured from the time measurement criteria are first met 
for CR until the first date that recurrent disease is objectively documented.
Duration of Stable Disease
Stable disease is measured from the start of the treatment until the criteria for progression 
are met, taking as reference the smallest measurements recorded since the treatment 
started. 
Response Review
This is performed by the Response Review Committee at the Roswell Park Cancer 
Institute.
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18. Ethical and Regulatory Standards
18.1
This study will not be initiated until the protocol and informed consent document(s) have 
been reviewed and approved by a properly constituted Institutional Review Board (IRB) 
or Independent Ethics Committee (IEC). Each patient (or legal guardian) shall read, 
understand, and sign an instrument of informed consent prior to performance of any 
study-specific procedure. It is the responsibility of the investigator to ensure that the 
patient is made aware of the investigational nature of the treatment and that informed 
consent is given.
The Investigator is responsible for the retention of the patient log and patient records; 
although personal information may be reviewed by authorized persons, that information 
will be treated as strictly confidential and will not be made publicly available. The 
investigator is also responsible for obtaining patient authorization to access medical 
records and other applicable study specific information according to Health Insurance 
Portability and Accountability Act regulations (where applicable).
This study will be conducted in compliance with all applicable laws and regulations of 
the state and/or country and institution where the patient is treated, in accordance with the 
Declaration of Helsinki, Good Clinical Practice, and according to the guidelines in this 
protocol, including attached appendices.
18.2
Informed Consent
The Investigator is responsible for obtaining written consent from each patient or the 
patient's legally authorized representative in accordance with ICH-GCP guidelines using 
the approved informed consent form, before any study specific procedures (including 
screening procedures) are performed. The informed consent form acknowledges all 
information that must be given to the patient according to ICH-GCP, including the 
purpose and nature of the study, the expected efficacy and possible side effects of the 
treatment(s), and specifying that refusal to participate will not influence further options 
for therapy. Any additional information that is applicable to the study must also be 
included. Additional national or institutionally mandated requirements for informed 
consent must also be adhered to. The patient should also be made aware that by signing 
the consent form, processing of sensitive clinical trial data and transfer to other countries 
for further processing is allowed.
The Investigator shall provide a copy of the information sheet and of the signed consent 
form to the patient and the signed original shall be maintained in the Investigator File. A 
copy of the signed consent form must be filed in the patient file. At any stage, the patient 
may withdraw from the study and such a decision will not affect any further treatment 
options.
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19. Study Responsibilities
19.1
Data Collection
Data entry into the database is to be completed in a timely fashion (approximately within 
28 days) after the patient’s clinic visit. If an adverse event is considered serious it is 
captured on both the Adverse Event page and the Serious Adverse Event Form, which is 
handled in an expedited fashion (see Section 16)
Data management activities will be performed using eRT.  eRT is a suite of software 
tools that enables the collection, cleaning and viewing of clinical trial data. CRS data 
management will design the study-specific database and facilitate it’s development by the 
eRT Information Technology team.  Once the database design is approved by the PI, 
Statistician, and Clinical Research Coordinator, the database will be put into production 
and data entry can begin.  Data can be entered and changed only by those with the rights 
to do so into the eCRFs (electronic CRFs). 
eRT is compliant with all relevant technical aspects of relevant GCP guidelines. 
•
The system can generate accurate copies of stored data and audit trail information 
in human readable form 
•
System access is limited to authorized individuals through the controlled 
assignment of unique ID and password combinations
•
The system is designed to periodically force users to change their passwords and 
verifies that user ID and password combinations remain unique. 
•
The system automatically generates a permanent time-stamped audit trail of all 
user interactions.
When data entry is complete, data management will review the data and will query any 
missing, incomplete, or invalid data points for resolution by the CRC and PI.  Once all 
queries have been resolved, the data can be released to the statistician for analysis. 
20. Maintenance of Study Documents
Essential documents should be retained per RPCI policy for 6 years from the study 
termination date. These documents could be retained for a longer period, however, if 
required by the applicable local regulatory requirements or by an agreement with RPCI. It 
is the responsibility of RPCI to inform the investigator/ institution as to when these 
documents no longer need to be retained. If, for any reason, the Investigator desires to no 
longer maintain the study records, they may be transferred to another institution, another 
investigator, or to RPCI upon written agreement between the Investigator and RPCI.
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21. Administrative Rules
21.1
Revisions to the Protocol
RPCI may make such changes to the protocol as it deems necessary for safety reasons or 
as may be required by the U.S. FDA or other regulatory agencies. Revisions will be 
submitted to the IRB/ERC for written approval before implementation.
21.2
Termination of the Study
It is agreed that, for reasonable cause, either the Investigators or the Sponsor, RPCI may 
terminate this study, provided a written notice is submitted within the time period 
provided for in the Clinical Trial Agreement. In addition, RPCI may terminate the study 
at any time upon immediate notice if it believes termination is necessary for the safety of 
patients enrolled in the study.
21.3 
Confidentiality
All information provided to the Investigator by RPCI including preclinical data, 
protocols, CRFs, and verbal and written information, will be kept strictly confidential and 
confined to the clinical personnel involved in conducting this study, and no disclosure 
shall be made except in accordance with any right of publication granted to the 
Investigator. This information may be related in confidence to the IRB/ERC or other 
committee functioning in a similar capacity. No report or information about the study will 
be provided to anyone not involved in the study without consent of RPCI except if 
required by law.
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Kepner JL, Chang MN. Samples of exact k-stage group sequential designs for Phase II 
and Pilot studies. Controlled clinical trials 2004;25:326-33.
55.
Shih C, Chen VJ, Gossett LS, et al. LY231514, a pyrrolo[2,3-d]pyrimidine-based 
antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 1997;57:1116-23.
56.
Rhee MS, Ryan TJ, Galivan J. Glutamyl hydrolase and the multitargeted antifolate 
LY231514. Cancer Chemother Pharmacol 1999;44:427-32.
57.
Chattopadhyay S, Zhao R, Krupenko SA, Krupenko N, Goldman ID. The inverse 
relationship between reduced folate carrier function and pemetrexed activity in a human colon 
cancer cell line. Molecular cancer therapeutics 2006;5:438-49.
58.
Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes 
small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-54.
59.
Jay C, Nemunaitis J, Chen P, Fulgham P, Tong AW. miRNA profiling for diagnosis and 
prognosis of human cancer. DNA and cell biology 2007;26:293-300.
60.
Leil TA, Endo C, Adjei AA, et al. Identification and characterization of genetic variation 
in the folylpolyglutamate synthase gene. Cancer Res 2007;67:8772-82.
61.
Ma CX, Adjei AA, Salavaggione OE, et al. Human aromatase: gene resequencing and 
functional genomics. Cancer Res 2005;65:11071-82.
62.
Adjei AA, Dy GK, Mandrekar SJ, et al. Pemetrexed pharmacogenomics: haplotype 
analyses of transport and activation/inactivation gene polymorphisms and the therapeutic 
outcome in non-small cell lung cancer, an NCCTG N0026 based study: PD2-3-5. Journal of 
Thoracic Oncology World Conference on Lung Cancer, Seoul, Korea, September 2-6, 2007 
2007;2:S445.
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63.
Adjei AA, Mandrekar SJ, Dy GK, et al. Phase II Trial of Pemetrexed Plus Bevacizumab 
for Second-Line Therapy of Patients With Advanced Non-Small-Cell Lung Cancer: NCCTG and 
SWOG Study N0426. 2009:JCO.2009.23.6406.
64.
Inoue S, Hashiguchi M, Takagi K, Kawai S, Mochizuki M. Preliminary study to identify 
the predictive factors for the response to methotrexate therapy in patients with rheumatoid 
arthritis. Yakugaku Zasshi 2009;129:843-9.
65.
Patino-Garcia A, Zalacain M, Marrodan L, San-Julian M, Sierrasesumaga L. 
Methotrexate in pediatric osteosarcoma: response and toxicity in relation to genetic 
polymorphisms and dihydrofolate reductase and reduced folate carrier 1 expression. J Pediatr 
2009;154:688-93.
66.
Ranganathan P, Culverhouse R, Marsh S, et al. Methotrexate (MTX) pathway gene 
polymorphisms and their effects on MTX toxicity in Caucasian and African American patients 
with rheumatoid arthritis. J Rheumatol 2008;35:572-9.
67.
Dulucq S, St-Onge G, Gagne V, et al. DNA variants in the dihydrofolate reductase gene 
and outcome in childhood ALL. Blood 2008;111:3692-700.
68.
Wessels JA, de Vries-Bouwstra JK, Heijmans BT, et al. Efficacy and toxicity of 
methotrexate in early rheumatoid arthritis are associated with single-nucleotide polymorphisms 
in genes coding for folate pathway enzymes. Arthritis Rheum 2006;54:1087-95.
69.
Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and 
haplotype maps. Bioinformatics 2005;21:263-5.
70.
Klee EW, Erdogan S, Tillmans L, et al. Impact of sample acquisition and linear 
amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-
dissection cell-sampling versus bulk tissue-sampling. BMC medical genomics 2009;2:13.
71.
Blower PE, Verducci JS, Lin S, et al. MicroRNA expression profiles for the NCI-60 
cancer cell panel. Molecular cancer therapeutics 2007;6:1483-91.
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Appendix: 1: Diary for Folic acid medication 
PATIENT INSTRUCTIONS:  Use this calendar to record your daily dose of Folic Acid
 Take 1 tablet  daily      
Patient Initials (Last, F, M)  
   
   
          Patient MR #:____________ 
Time Period From:  
   /  
   /   
                         
         To:  
   /   
   /   
M  M             D   D              Y     Y    Y   Y                                                    M  M         D   D           Y    Y   Y    Y
Notes:
Be sure to bring this calendar and the pill bottle with you for all of your return appointments.
Return the calendar and the pill bottle to your Clinical Research Coordinator.  Thank you.
DAY  Of WEEK
Sunday 
DAY  OF WEEK
Monday 
DAY  OF WEEK
Tuesday
DAY  OF WEEK
Wednesday
DAY  OF WEEK
Thursday
DAY  OF WEEK
Friday
DAY  OF WEEK
Saturday  
Day of cycle
# of tablets taken:
AM ________
PM ________
Day of cycle
# of tablets taken:
AM ________
PM ________
Day of cycle
 
# of tablets taken:
AM ________
PM ________
Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
Day of cycle
# of tablets taken:
AM________
PM________
 Day of cycle
 
# of tablets taken:
AM_________
PM_________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
Day of cycle
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
Day of cycle
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________
 Day of cycle
 
# of tablets taken:
AM ________
PM ________


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Appendix 2: Network Sites
1. CONTACT INFORMATION
All questions related to the protocol or study implementation should be directed to:
Roswell Park Cancer Institute
CRS Network Office
ASB K 102B
Buffalo, New York 14263
Telephone:
716-845-8084 or 716-845-1203 - M-F; 7:00 AM to 4:30 PM 
716-845-2300 - After hours, weekends and holidays: request the RPCI Principal Investigator
Fax: 
716-845-8743
2. INFORMED CONSENT
•
Informed Consent must be obtained by the Investigator from any patients wishing to 
participate, prior to any procedures or change in their treatment.
•
An informed consent template is provided by RPCI and can be amended to reflect 
institutional requirements.
•
All consent changes must be reviewed by Roswell Park Cancer Institute Network Office 
prior to submission to the site IRB.
•
The informed consent must be IRB approved.
•
Always check that you are using the correct date and version of the IRB approved 
consent.
3. SUBJECT REGISTRATION 
Phase II protocol registration instructions:
The Subject Enrollment Log must be faxed to the CRS Network Office within 24 hours of 
the date the patient is consented. Once the Principal Investigator has determined that 
eligibility has been met, complete the Subject Registration Form and fax it to the RPCI 
Network Office at (716) 845-8743.  
Note: The subject completes the Gender, Race, and Ethnicity form and this is placed in the 
study binder.
Roswell Park Cancer Institute does not grant exceptions to eligibility criteria.
4. STUDY DEVIATIONS
•
If a deviation has occurred to eliminate hazard, this should be reported to the RPCI 
Network, site IRB and any other regulatory authority involved in the trial.
•
ANY study deviation will be recorded on the Study Deviation Log.
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•
Subjects who are inadvertently enrolled, with significant deviation(s) from the study-
specified criteria, will be removed from the study.
•
Notify RPCI of any early subject withdrawal and appropriately document the 
discontinuation and the reason why.
5. STUDY DOCUMENTATION
•
Study documents must be filled out completely and correctly. Ditto marks are not 
allowed.
•
If an entry has been documented in error put a single line through the entry and initial and 
date the change. The auditor must be able to read what has been deleted. 
o
Do NOT use white-out, magic marker, scratch-outs.
o
Do NOT erase entries. 
•
Use only black ink for documentation on the accountability form and any other study 
forms.
6. DRUG ACCOUNTABILITY
Drug accountability will be strictly maintained by recording quantities of study drug 
received, dispensed to patients and wasted, lot number, date dispensed, patient ID number 
and initials, quantity returned, balance remaining, manufacturer, expiration date, and the 
initials of the person dispensing the medication. 
•
Responsibility rests solely with the Principal Investigator but can be delegated as 
appropriate (e.g. to pharmacy personnel).
•
Records must be maintained regarding receipt, dispensing, return, waste and disposition 
of all investigational agents.
•
Study drug supply should only be used in accordance with the IRB approved study.
•
Drug accountability forms are protocol and agent specific, they are study source 
documents and will be used to verify compliance with the study.
•
Any discrepancies shall be documented and explained.
•
An inventory count should be performed with each transaction.
•
Drug accountability forms shall be stored with study related documents.
•
Each medication provided for this study and each dosage form and strength must have its 
own Drug accountability. 
•
Do NOT “transfer”, “borrow” or “replace” supplies between studies.
•
Dispensing the wrong study supply is considered a medication error.
•
Never replace investigational agents with commercial product.
7. SERIOUS ADVERSE EVENT REPORTING:
The Investigator will utilize the MedWatch Form for the reporting of adverse events and 
follow up information to those events.
The site Investigator or designated research personnel will report all serious adverse events, 
whether related or unrelated to the investigational agent(s) to the IRB in accordance with 
their local institutional guidelines. 
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All serious adverse events regardless of severity or relationship must be reported to Allos 
within 24 hours of the investigational staff’s knowledge.  
“Spectrum Pharmaceuticals
157 Technology Drive
Irvine, CA 92618
Phone: (949) 788-6700
Fax: (949) 788-6706; be sure to retain successful fax acknowledgement
Email:  drugsafety@sppirx.com” 
And
NCCN via fax at 215-358-7699 or e-mailed to ORPReports@nccn.org
The site will notify the CRS Network Office within one business day of being made aware of 
the SAE. A preliminary written report must follow within 24 hours (1 business day) of the 
oral notification using the following forms:
•
SAE report form
•
MedWatch 3500A 
A complete follow-up report must be filed within 10 working days
8. UNANTICIPATED PROBLEM REPORTING:
An Unanticipated Problem (UAP) is any incident, experience, or outcome that meets all of 
the following criteria:
1. Unexpected (in terms of nature, severity, or frequency) given:
(a) the research procedures that are described in the study- related documents, 
including study deviations, as well as issues related to compromise of patient 
privacy or confidentiality of data; 
(b) the characteristics of the subject population being studied;
2. Related or possibly related to participation in the research (possibly related means 
there is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research);
3. Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized;
For all adverse events occurring that are unanticipated and related or possibly related to the 
research drug, biologic or intervention the participating physician or delegated research staff 
from each site will notify their local IRB in accordance with their local institutional 
guidelines.  The site must also notify the CRS Network Office within 24 hours of being 
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made aware of the Unanticipated Problem by completing the RPCI Unanticipated Problem 
Report Form and faxing it to the CRS Network office.
9. SAMPLE SHIPPING INSTRUCTIONS
MicroRNA

Formalin-fixed paraffin embedded tissue block   or 5-10 unstained formalin-fixed 
paraffin embedded sections on appropriate slides and one Hematoxylin and Eosin stained 
slide of the primary tumor

Labeled with Patient initials, Subject ID (provided by RPCI), Protocol number I 169210 
and date and time of collection

Shipment Notification email to  Eric.Kannisto@roswellpark.org 

Ship samples overnight  (Monday through Thursday)
SHIPPING ADDRESS:
Roswell Park Cancer Institute
Eric Kannisto/ Dr. Yendamuri
Medical Research Complex (MRC) - Room 207 
RE: I 169210 Specimens
Elm & Carlton Streets
Buffalo, NY 14263
716-845-8364
Genotype DNA Samples:
•
After collection in EDTA tubes, blood sample should be decanted into 5 mL cryovials 
and stored at -70 to -80 C
•
Labeled with Patient initials, Subject ID (provided by RPCI), Protocol number I 
169210 and date and time of collection
•
Shipment Notification:  Huayi.huang@RoswellPark.org
•
Shipment frozen aliquots overnight on dry ice (Monday through Thursday)
SHIPPING ADDRESS:
Roswell Park Cancer Institute
RE: I 169210 PG Specimens 
Attn: Huang Huayi ( c/o Dr. Araba Adjei)
Dept of Pharmacology & Therapeutics 
Grace Cancer Drug Center Room G127
Elm & Carlton Streets
Buffalo, New York 14263
(716) 845-7656 
APPROVED RPCI Institutional Review Board: 5/5/2017