Conditions: Recurrent Plasma Cell Myeloma Refractory Plasma Cell Myeloma
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 12
Healthy volunteers: no

Brief summary:
This phase I/II trial studies the side effects and best dose of leflunomide in treating patients with multiple myeloma that has come back (relapsed) or has not responded to previous treatment (refractory). Leflunomide may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth.

Detailed description:
PRIMARY OBJECTIVES: I. To determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of leflunomide, when given as a single agent. (Phase I) II. To assess the safety and tolerability of leflunomide at each dose level by evaluation of toxicities including: type, frequency, severity, attribution, time course and duration. (Phase I) III. To evaluate the anti-myeloma activity of leflunomide, when given as a single agent, as assessed by overall response rate (ORR). (Phase II)

SECONDARY OBJECTIVES: I. To obtain estimates of: response duration, clinical benefit response, overall survival, progression-free survival. (Phase II)

TERTIARY OBJECTIVES: I. To characterize the relationship between serum concentration of the active leflunomide metabolite, teriflunomide (A77 1726), and toxicity. (Phase I/II) II. To assess the relationship between serum concentration of the active leflunomide metabolite, teriflunomide (A77 1726), and disease response. (Phase I/II) III. To explore the relationship between polymorphisms in the CYP1A2, CYP2C19, or DHODH genes and toxicity/response. (Phase I/II) IV. To explore the ex vivo cytotoxicity of leflunomide toward primary multiple myeloma (MM) cells, in order to evaluate whether individual ex vivo leflunomide response might be a useful predictor of therapeutic response. (Phase I/II) V. To explore the potential additive or synergistic effects of combining leflunomide with other classes of Food and Drug Administration (FDA)-approved drugs. (Phase I/II) VI. To generate a preliminary ribonucleic acid (RNA)/microRNA (miRNA) and deoxyribonucleic acid (DNA) methylation signature associated with response of MM cells to leflunomide in vivo (mRNA/miRNA and DNA methylation, phase II only) and teriflunomide ex vivo (messenger RNA \[mRNA\]/miRNA). (Phase I/II)

OUTLINE: This is a dose-escalation study. Patients receive leflunomide orally (PO) once daily (QD) on days 1-28. Courses repeat every 28 days in the absence of disease progression or unacceptable toxicity. After completion of study treatment, patients are followed up at 30 days and then every 28 days until disease progression (active follow-up) or every 3 months (long term follow-up).

Arms:
arm 1: Patients receive 20 mg leflunomide PO QD on days 1-28. Courses repeat every 28 days in the absence of disease progression or unacceptable toxicity. arm 2: Patients receive 40 mg leflunomide PO QD on days 1-28. Courses repeat every 28 days in the absence of disease progression or unacceptable toxicity. arm 3: Patients receive 60 mg leflunomide PO QD on days 1-28. Courses repeat every 28 days in the absence of disease progression or unacceptable toxicity.

Interventions: intervention 1: Laboratory Biomarker Analysis intervention 2: Leflunomide intervention 3: Pharmacological Study
Intervention descriptions: intervention 1: Correlative studies intervention 2: Given PO intervention 3: Correlative studies

Protocol document:
Prot_SAP_000.pdf: 
IRB Protocol No. 15140 
 
Version Date:  08/01/19               
Version: 09 
CITY OF HOPE NATIONAL MEDICAL CENTER 
1500 E. DUARTE ROAD 
DUARTE, CA 91010 
DEPARTMENT OF HEMATOLOGY AND HEMATOPOIETIC CELL TRANSPLANTATION 
 
TITLE:  A Phase I/II Dose-Escalation Trial of Leflunomide in Patients with Relapsed or 
Relapsed/Refractory Multiple Myeloma  
 
CITY OF HOPE PROTOCOL 
NUMBER: 
15140 
VERSION: 
09 
COH Initial Submission Protocol dated 09/02/2015  
 
 
 
 
   00 
COH Amendment 01 Protocol dated 09/17/2015 
 
 
 
 
 
   01 
COH Amendment 02 Protocol dated 11/05/2015 
 
 
 
 
 
   02 
COH Amendment 03 Protocol dated 06/13/2016 
 
 
 
 
 
   03 
COH Amendment 04 Protocol dated 11/09/2016                                                                    04   
COH Amendment 05 Protocol dated 04/14/2017                                                                    05 
COH Amendment 06 Title Page dated 06/20/2017                                                     
   06    
COH Amendment 07 Protocol dated 10/23/2017 
 
 
 
 
 
   07 
COH Amendment 08 Title Page dated 03/11/2019                                                    
   08 
COH Amendment 09 Title Page at Continuation dated 08/01/19 
 
 
 
   09    
 
SPONSOR/IND NUMBER:   
City of Hope/127341 
DISEASE SITE: 
 
Multiple Myeloma  
STAGE (if applicable): 
 
Refractory or Relapsed  
MODALITY: 
 
Oral 
PHASE/TYPE: 
 
Phase I/II- Intervention 
 
PRINCIPAL INVESTIGATOR: 
Michael A. Rosenzweig, MD 
CO- INVESTIGATOR(S): 
 
Amrita Krishnan, MD 
Joycelynne M. Palmer, PhD 
Steven Rosen, MD 
Tim Synold, PharmD 
 
PARTICIPATING CLINICIANS: 
 
Len Farol, M.D., Alex Herrera, M.D., Myo Htut, M.D.,  
Chatchada Karanes, M.D., Auayporn Nademanee, 
M.D., Nitya Nathwani, M.D., Firoozeh Sahebi, M.D., 
Tanya Siddiqi, M.D.,  
 
 
STUDY STATISTICIAN: 
Joycelynne M. Palmer, PhD, Arnab Chowdhury, M.D. 
PARTICIPATING SITES: 
City of Hope  
 


Page 1 of 59 
IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
 
 
 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 91010 
Clinical Trial Protocol 
 
A Phase I/II Dose-Escalation Trial of Leflunomide in Patients with  
Relapsed or Relapsed/Refractory Multiple Myeloma 
 
Protocol Version Date:   
10/23/2017 
Protocol Version No.:   
05 
COH Protocol No.: 
 
15140 
Agent:   
 
 
Leflunomide, commercially available 
IND#:   
 
 
127341 
IND holder: 
 
 
City of Hope 
Coordinating Center:   
City of Hope (dcc@coh.org) 
 
Principal Investigator 
Michael A. Rosenzweig, MD 
Hematologic Malignancies and Stem 
Cell Transplantation Institute 
City of Hope National Medical Center 
T: 626-256-4673 ext 62405 
Email: mrosenzweig@coh.org 
 
Biostatistician/Co-Investigator 
Joycelynne M. Palmer, PhD 
Hematologic Malignancies and Stem 
Cell Transplantation Institute 
City of Hope National Medical Center 
T: 626-256-4673 ext 65266 
Email: jmpalmer@coh.org 
 
 
Co-Investigator 
Tim Synold, PharmD 
Department of Cancer Biology 
City of Hope Beckman Research 
Institute 
T: 626-256-4673 ext 62110 
Email: tsynold@coh.org 
 
Co-Investigator 
Amrita Krishnan, MD 
Director, Multiple Myeloma Program 
Hematologic Malignancies and Stem 
Cell Transplantation Institute 
City of Hope National Medical Center 
T: 626-256-4673 ext 62405 
Email: akrishnan@coh.org 
 
Co-Investigator 
Steven T. Rosen, MD 
Hematologic Malignancies and Stem 
Cell Transplantation Institute 
City of Hope National Medical Center 
and Beckman Research Institute 
T: 626-256-4673 ext 67330 
Email: srosen@coh.org 
 
 


Phase I/II: Leflunomide for Multiple Myeloma 
Page 2 of 59 
IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
EXPERIMENTAL DESIGN SCHEMA 
 
*** Blood for research studies will be taken at the time of regular clinical labs. Bone marrow aspirate will be 
collected on the first day of cycle 2 from all phase II patients and phase I patients enrolled at the anticipated 
RP2D. Leftover bone marrow aspirate samples from clinical procedures will also be used for research. 


Phase I/II: Leflunomide for Multiple Myeloma 
Page 3 of 59 
IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
PROTOCOL SYNOPSIS 
Protocol Title: 
A phase I/II dose-escalation trial of leflunomide in patients with relapsed or relapsed/refractory 
multiple myeloma 
Brief Protocol Title for the Lay Public (if applicable): 
Leflunomide in relapsed or refractory multiple myeloma 
Study Phase: 
Phase I/II 
Participating Sites: 
Single Center: City of Hope, Duarte 
Rationale for this Study: 
Despite many advances in treatment, multiple myeloma remains an incurable disease and new, 
effective treatments are needed. Leflunomide is a commercially available oral immunosuppressive 
agent that has been FDA approved since 1998 for the treatment of rheumatoid arthritis (RA). The 
primary mechanism of action of leflunomide is inhibition of de novo pyrimidine synthesis by 
targeting dihydroorotate dehydrogenase (DHODH), and thus achieving anti-proliferative effect in B- 
and T-lymphocytes. Leflunomide has been investigated for its anti-neoplastic potential in a variety of 
pre-clinical tumor models. Recent pre-clinical studies have demonstrated significant anti-neoplastic 
activity in multiple myeloma. In addition, limited clinical experience is suggestive of a beneficial 
response when leflunomide is used to treat relapsed/refractory multiple myeloma. Considering the 
favorable toxicity profile and extensive clinical experience with leflunomide in rheumatoid arthritis, 
this drug represents a potential new candidate for therapy in multiple myeloma.  
Objectives: 
In patients with relapsed or relapsed/refractory multiple myeloma: 
Phase I 
Primary Objectives: 
o 
To determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) 
of leflunomide, when given as a single agent. 
o 
To assess the safety and tolerability of leflunomide at each dose level by evaluation of 
toxicities including: type, frequency, severity, attribution, time course and duration. 
 
Phase II 
Primary Objective: 
o To evaluate the anti-myeloma activity of leflunomide, when given as a single agent, as 
assessed by overall response rate (ORR). 
Secondary Objectives: 
o 
To obtain estimates of:  


Phase I/II: Leflunomide for Multiple Myeloma 
Page 4 of 59 
IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
o 
Response duration 
o 
Clinical benefit response 
o 
Overall survival and  
o 
Progression-free survival. 
 
Clinical Pharmacology Objectives (Phase I and II) 
o 
To characterize the relationship between serum concentration of the active leflunomide 
metabolite, teriflunomide (A77 1726), and toxicity. 
o 
To assess the relationship between serum concentration of the active leflunomide 
metabolite, teriflunomide A77 1726), and disease response.  
Exploratory Ex-Vivo and Molecular Objectives (Phase I and II) 
o 
To explore the relationship between polymorphisms in the CYP1A2, CYP2C19, or DHODH 
genes and toxicity/response. CYP1A2, CYP2C19 are the hepatic enzymes responsible for 
leflunomide metabolism. 
o To explore the ex vivo cytotoxicity of leflunomide toward primary MM cells, in order to 
evaluate whether individual ex vivo leflunomide response might be a useful predictor of 
therapeutic response.  
o To explore the potential additive or synergistic effects of combining leflunomide with 
other classes of FDA-approved drugs. The classes include HDAC inhibitors, 
immunomodulatory drugs [IMiDs], proteasome inhibitors, steroid hormones, and 
antibody therapeutics such as the CD38 antibody daratumumab. 
o To generate a preliminary RNA/miRNA and DNA-methylation signature associated with 
response of MM cells to leflunomide in vivo and teriflunomide ex vivo. 
 
Study Design: 
This study will be conducted as a single center, single agent phase I/II trial.  
The phase I portion will implement a modified rolling six dose escalation design, a more conservative 
version of the rolling six design of Skolnik, et al. [1], for enrollment with dose escalation, de-
escalation, or expansion of a cohort on the basis of the occurrence of dose limiting toxicities (DLTs) 
during cycle 1.  
The starting dose of leflunomide is 20mg daily, with the intention to test up to 60mg daily. Escalation 
will proceed in increments of 20mg/day (odd numbered dose levels); de-escalation will proceed in 
decrements of 10mg/day. Up to six doses of leflunomide will be considered:  
Dose Level 
Leflunomide Dose* 
-1 
10 mg PO daily 
1 
20 mg PO daily 
2 
30 mg PO daily 
3 
40 mg PO daily 
4 
50 mg PO daily 
5 
60 mg PO daily 
*NOTE: Leflunomide is administered with a loading dose of 100 mg for the 1st three doses. 


Phase I/II: Leflunomide for Multiple Myeloma 
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IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
The highest dose level that produces ≤1/6 DLTs in cycle 1 will be the MTD. The RP2D of leflunomide 
will generally be the MTD, but it may be less than the MTD based on a review of available 
data/cumulative toxicities.  
Following the phase I trial, a single arm phase II trial will be conducted using a Simon Two-Stage 
Optimal Design [2, 3] to evaluate the anti-myeloma activity of leflunomide, when given as a 
single agent, as assessed by overall response rate (ORR). The phase II portion of the study is 
expected to enroll a minimum of 19 and a maximum of 39 patients. The sample size is based on 
the desire to discriminate a promising ORR of 30% from a disappointing response rate of 15% 
using a type I error rate of 0.10 and power of 80%. 
Patients will be treated in planned 28-day treatment cycles until disease progression, unacceptable 
toxicity, or other criteria for removal from study treatment are satisfied. Patients will undergo 
monthly evaluation for response using the International Myeloma Working Group (IMWG) criteria.  
Endpoints: 
Phase I: The primary endpoint is toxicity. Toxicity will be graded according to the NCI-Common 
Terminology Criteria for Adverse Events version 4.03. Dose limiting toxicity (DLT) is defined in Section 
13.1 of the protocol. 
Phase II: The primary endpoint is response. Response will be categorized using the IMWG response 
criteria for multiple myeloma [37, 38]. The response categories and criteria are summarized in Table 
11.2 of the protocol. 
Sample Size: 
The expected phase I sample size is 12-18 patients; allowing for 6 patients to replace 
inevaluable/ineligible patients. The expected phase II sample size is 19-39 patients. It is anticipated 
that the 6 patients from the phase I portion who are treated at the R2PD will be included in the 
sample size for the phase II portion (phase I/II participants).  
Estimated Duration of the Study 
With an expected accrual rate of approximately 1 patient enrolled per month, the phase I portion of 
the trial is expected to complete accrual in 12-18 months. The phase II portion is expected to accrue 
2 patients per month, with completion in 17 months. The expected maximum duration of therapy is 
24 months. The total study is expected to last a maximum of (18+17+24) 59 months or 
approximately 5 years.  
Summary of Subject Eligibility Criteria: 
Inclusion Criteria: 
Participants must meet the following criteria on screening examination to be eligible to participate in 
the study: 
 
All subjects must have the ability to understand and the willingness to sign a written 
informed consent. 
 
Patients must be age ≥ 18 years. 
 
Patients must have a life expectancy of > 3 months. 
 
Patients must exhibit an ECOG performance status of 0-2. 
 
Patients must have a diagnosis of multiple myeloma. 
 
Patients must have measureable disease, defined as one of the following within 21 days 
prior to registration: 
o 
Serum M-protein ≥ 0.5 g/dL 
o 
Urine M-protein ≥ 200 mg/24 hr 


Phase I/II: Leflunomide for Multiple Myeloma 
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IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
o 
Serum free light chain ≥ 10 mg/dL provided the FLC ratio is abnormal. 
o 
10% plasma cells in bone marrow 
 
Patients must be relapsed or are refractory to at least 3 prior lines of therapy, including both 
a proteasome inhibitor and an IMiD, and for whom transplant is not recommended 
(induction therapy and stem cell transplant +/- maintenance will be considered as one 
regimen). 
 
At least 2 weeks from prior therapy to time of start of treatment. Prior therapy includes 
steroids (except prednisone or equivalent – up to 10 mg per day is allowed). 
 
Platelet count > 50,000/µL. Platelet transfusions are not allowed within 14 days of platelet 
assessment.  
 
ANC ≥ 1000/mm3. Growth factor is not permitted within 14 days of neutrophil assessment. 
 
AST and ALT < 2.0 x ULN. 
 
Total Bilirubin < 1.5 x ULN. 
 
Calculated creatinine clearance (CrCl) ≥ 30 mL/min per 24-hour urine collection or the 
Cockcroft-Gault formula. 
 
Negative serum or urine β-HCG test (female patient of childbearing potential* only), to be 
performed locally within the screening period.  
 
Negative for tuberculosis antigen (e.g. T-Spot test). 
 
Negative for hepatitis A, B, or C infection.  
 
Adequate pulmonary function as defined by FVC and DLCO ≥ 50% of predicted by pulmonary 
function testing.  
 
Agreement by females of childbearing potential* and sexually active males to use an 
effective method of contraception (hormonal or barrier method of birth control or 
abstinence) prior to study entry and for three months following duration of study 
participation. The effects of study treatment on a developing fetus have the potential for 
teratogenic or abortifacient effects. Should a woman become pregnant or suspect that she is 
pregnant while participating on the trial, she should inform her treating physician 
immediately. *A female of childbearing potential is defined as a sexually mature woman 
who: 1) has not undergone a hysterectomy or bilateral oophorectomy; or 2) has not been 
naturally postmenopausal for at least 24 consecutive months. 
Exclusion Criteria  
Prospective participants who meet any of the following criteria will not be eligible for admission into 
the study: 
 
Prior treatment with leflunomide. 
 
Current or planned use of other investigational agents, or concurrent biological, 
chemotherapy, or radiation therapy during the study treatment period. (See inclusion 
criterion #8 for washout times). 
 
Current or planned growth factor or transfusion support until after initiation of treatment. If 
growth factor or transfusion support is provided between screening and start of treatment, 
the participant will no longer be eligible.  
 
Prior diagnosis of rheumatoid arthritis. 
 
Prior Allogeneic transplant. 
 
Acute active infection requiring systemic therapy within 2 weeks prior to enrollment. 
 
Pre-existing liver disease.  
 
Known HIV infection. 
 
History of allergic reactions attributed to compounds of similar chemical or biologic 


Phase I/II: Leflunomide for Multiple Myeloma 
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IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
composition to leflunomide or cholestyramine. 
 
Non-hematologic malignancy within the past 3 years aside from the following exceptions:  
o 
adequately treated basal cell or squamous cell skin cancer 
o 
carcinoma in situ of the cervix 
o 
prostate cancer < Gleason Grade 6 with a stable PSA 
o 
successfully treated in situ carcinoma of the breast 
 
Clinically significant medical disease or condition that, in the investigator’s opinion, may 
interfere with protocol adherence or the patient’s ability to give informed consent.  
 
Pregnant women and women who are lactating. Leflunomide has potential for teratogenic 
or abortifacient effects. Because there is a potential risk for adverse events in nursing infants 
secondary to treatment of the mother with these agents, breastfeeding should be 
discontinued if the mother is enrolled on this study.  
 
Any other condition that would, in the Investigator’s judgment, contraindicate the patient’s 
participation in the clinical study due to safety concerns or compliance with clinical study 
procedures, e.g., infection/inflammation, intestinal obstruction, unable to swallow 
medication, social/ psychological issues, etc. 
 
Prospective participants who, in the opinion of the investigator, may not be able to comply 
with all study procedures (including compliance issues related to feasibility/logistics).  
 
Investigational Product Dosage and Administration: 
Leflunomide will be administered orally at an initial dose of 20 mg daily following a loading dose of 
100 mg per day for the first three days of administration. For the phase I portion of the study, five 
daily dose levels of leflunomide may be tested (10, 20, 30, 40 or 60 mg daily) There will be no intra-
patient dose escalation. The R2PD will be used as the daily dose for the phase II portion of the study. 
At the end of the study, all participants will receive 11 days of oral cholestyramine treatment (8 g 
three times daily) to reduce the plasma leflunomide levels.  
Clinical Observations and Tests to be Performed: 
Clinical observations will include tolerance as well as response to treatment. Baseline studies will 
include: history and physical, pulmonary function test, skeletal survey and bone marrow aspiration 
and biopsy (the bone marrow aspiration will be used for clinical assessment and leftover samples will 
be used for correlative laboratory studies), as well as a laboratory evaluation to include CBC with 
differential, comprehensive chemistry panel (to include LDH, magnesium, phosphorus and uric acid) 
and myeloma labs (serum protein electrophoresis, serum immunofixation, 24-hour urine protein 
electrophoresis, urine immunofixation, serum free light chains, and quantitative immunoglobulins). 
Clinical observation of response will take place on day 1 of each 28-day cycle. Subjects will be 
evaluated through clinic visits and physical exams as well as laboratory tests to include CBC with 
differential, comprehensive chemistry panel and myeloma labs. Blood tests will be performed at a 
minimum of every 28 days but may be done more frequently as clinically indicated. A skeletal survey 
may be repeated as clinically indicated. Patients will undergo toxicity assessment every two weeks 
for the first three cycles following initiation with leflunomide and will undergo, at a minimum, 
monthly toxicity assessments thereafter. For all phase II patients and phase I patients enrolled at the 
anticipated RP2D, a second bone marrow aspiration will be conducted for correlative laboratory 
studies on day 1 of cycle 2. Confirmation of complete response (CR), stringent complete response 
(sCR), and progressive disease (PD) will involve bone marrow aspiration and, for patients who have 
enrolled in the phase II portion, leftover samples from these bone marrow aspirations will be used 


Phase I/II: Leflunomide for Multiple Myeloma 
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IRB Protocol No. 15140 
 
Version Date:  10/23/17 
Version: 07 
for correlative laboratory studies.  
Statistical Considerations: 
Analysis: Observed toxicities will be summarized in terms of type (organ affected or laboratory 
determination), severity, attribution, time of onset, duration, serum concentration of the active 
leflunomide metabolite, probable association with the study treatment and reversibility or outcome. 
Baseline information (e.g. the extent/type of prior therapy) and demographic information will be 
presented as well to describe the patients treated in this study. 
Rates and 95% Clopper Pearson binomial confidence interval (CI) will be calculated for overall 
response rate (patients that have confirmed sCR/CR/VGPR or PR) and clinical benefit rate (patients 
that have confirmed sCR/CR/VGPR/PR/MR or SD). In addition, sub-analyses will be performed where 
participants will be considered evaluable for response if they are confirmed eligible, receive at least 
75% of leflunomide during the first cycle of therapy and have their disease re-evaluated. Response 
rates will also be explored based on number/type of prior therapy(ies) and serum concentration of 
the leflunomide metabolite (µg/mL). Time to response and survival will be estimated using the 
product-limit method of Kaplan and Meier. 
Serum trough levels of leflunomide’s active metabolite, teriflunomide (A77 1726), will be measured 
in each patient at multiple time points throughout the study. Both total and free teriflunomide will 
be tested. The association between serum levels and toxicity, as well as response, will be evaluated 
at the conclusion of the study.  
Genetic polymorphisms in genes associated with the biological activity of leflunomide (e.g., DHODH, 
CYP1A2, and CYP2C19) have been previously described and will be explored for possible associations 
with response and safety outcomes.  
Ex vivo studies will be performed on bone marrow aspirate-derived cells to evaluate the IC50 for 
teriflunomide and the potential synergy with other anti-neoplastic agents. Correlation analyses will 
be performed to determine the relationship between ex vivo and in vivo response.  
Preliminary molecular profiles for miRNA, mRNA, and DNA methylation status will be derived from 
both ex vivo and in vivo treatment. These profiles will be used to identify molecular correlates of 
therapeutic response using pathway identification and supervised principal components analysis. 
Sponsor/Licensee: 
Investigator Initiated: City of Hope 
Case Report Forms 
Medidata RAVE® Electronic Data Capture system. Forms are detailed in Section 12.1.3. 


Phase I/II: Leflunomide for Multiple Myeloma 
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TABLE OF CONTENTS 
SECTION ....................................................................................................................................................PAGE 
Experimental Design Schema ......................................................................................................................... 2 
Protocol Synopsis............................................................................................................................................ 3 
Exclusion Criteria .............................................................................................................................. 6 
Table of Contents ........................................................................................................................................... 9 
Abbreviations ................................................................................................................................................ 12 
1.0 
Goals and Objectives (Scientific Aims) ........................................................................................... 13 
2.0 
Background..................................................................................................................................... 14 
2.1 
Introduction/Rationale for Development ........................................................................ 14 
2.2 
Leflunomide ..................................................................................................................... 14 
2.3 
Preclinical Studies of Leflunomide as an Anti-Neoplastic Agent ...................................... 15 
2.4 
Clinical Experience with Leflunomide for Multiple Myeloma .......................................... 15 
2.5 
Background for Correlative Studies ................................................................................. 16 
2.6 
Overview and Rationale of Proposed Study..................................................................... 16 
3.0 
Patient Eligibility ............................................................................................................................. 18 
3.1 
Inclusion Criteria .............................................................................................................. 18 
3.2 
Exclusion Criteria .............................................................................................................. 19 
3.3 
Inclusion of Women and Minorities ................................................................................. 20 
4.0 
Screening and Registration Procedures ......................................................................................... 20 
4.1 
Screening Procedures ...................................................................................................... 20 
4.2 
Informed Consent ............................................................................................................ 21 
4.3 
Registration Requirements/Process ................................................................................ 21 
4.4 
Screen Failures and Registered Participants Who Do Not begin Study Treatment ......... 22 
5.0 
Treatment Program ........................................................................................................................ 22 
5.1 
Treatment Overview ........................................................................................................ 22 
5.2 
Treatment cycle definition ............................................................................................... 22 
5.3 
Phase I/II Treatment Plan ................................................................................................. 22 
Table 5.3.1: Dose Levels ................................................................................................................. 23 
5.4 
Phase II Treatment Plan ................................................................................................... 23 
5.5 
Agents Administration ..................................................................................................... 23 
5.6 
Study Procedures ............................................................................................................. 23 
5.7 
End of Treatment Evaluations and Leflunomide Detoxification ...................................... 23 
5.8 
Duration of Therapy & Criteria for Removal from Study Treatment ............................... 24 
5.9 
Active Follow Up and Long Term Follow Up .................................................................... 24 
5.10 
Criteria for Completion of Study Participation ................................................................. 25 
5.11 
Supportive Care, Other Concomitant Therapy, Prohibited Medications ......................... 25 
5.12 
Additional Studies ............................................................................................................ 25 
6.0 
Anticipated Toxicities and Dose Delays/Modifications for Adverse Events ................................... 25 
6.1 
Anticipated Toxicities ....................................................................................................... 25 
6.2 
Dose Modifications of Leflunomide ................................................................................. 27 
Table 6.2.2 Dose Modifications for Leflunomide Treatment ......................................................... 27 


Phase I/II: Leflunomide for Multiple Myeloma 
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6.3 
Dose Modifications of Cholestyramine ............................................................................ 30 
7.0 
Data and Safety Monitoring, Unanticipated Problem and Adverse Event Reporting .................... 30 
7.1 
Definition of Risk Level ..................................................................................................... 30 
7.2 
Monitoring and Personnel Responsible for Monitoring .................................................. 30 
7.3 
Dose Escalation ................................................................................................................ 31 
7.4 
Adverse Events and Serious Adverse Events.................................................................... 31 
7.5 
Adverse Event Name and Severity ................................................................................... 31 
7.6 
Adverse Event Attribution ................................................................................................ 32 
7.7 
COH Held IND ................................................................................................................... 32 
7.8 
Deviations and Unanticipated Problems .......................................................................... 32 
7.9 
Single Subject Exception (SSE) ......................................................................................... 33 
8.0 
Agent Information. ......................................................................................................................... 33 
8.1 
Leflunomide ..................................................................................................................... 33 
8.2 
Cholestyramine ................................................................................................................ 34 
9.0 
Correlative/Special Studies ............................................................................................................ 35 
9.1 
Pharmacokinetics ............................................................................................................. 35 
9.2 
Pharmacodynamics .......................................................................................................... 36 
9.3 
Pharmacogenomics .......................................................................................................... 37 
9.4 
Ex vivo and Molecular Studies ......................................................................................... 37 
Table 9.3.2 Distribution of CD138-enriched bone marrow aspirate cells for research .................. 39 
10.0 
Study Calendar ............................................................................................................................... 40 
Table 10.0 Study Activity Calendar ................................................................................................. 40 
11.0 
Evaluation Criteria/Measurement of Effect ................................................................................... 43 
11.1 
Phase I/Primary Endpoint: Toxicity .................................................................................. 43 
11.2 
Phase II/Primary Endpoint: Response .............................................................................. 43 
Table 11.2 Response Criteria .......................................................................................................... 43 
11.3 
Phase II/Secondary Endpoints: ........................................................................................ 44 
12.0 
Data Reporting/Protocol Deviations .............................................................................................. 45 
12.1 
Data Reporting ................................................................................................................. 45 
Table 12.1.3 Data Submission Schedule ......................................................................................... 45 
12.2 
Protocol Deviations .......................................................................................................... 46 
13.0 
Statistical Considerations ............................................................................................................... 47 
13.1 
Definition of Dose-Limiting Toxicity (DLT) ........................................................................ 47 
13.2 
Evaluable Participants and Participant Replacement ...................................................... 47 
13.3 
Study Design..................................................................................................................... 48 
Table 13.3.1 Dose Levels ................................................................................................................ 48 
Table 13.3.2: Dose Escalation Rules ............................................................................................... 49 
Phase II Design ............................................................................................................................... 49 
13.4 
Sample Size and Accrual Rate .......................................................................................... 50 
13.5 
Statistical Analysis Plan .................................................................................................... 50 
14.0 
Human Subject Issues..................................................................................................................... 52 
14.1 
Institutional Review Board ............................................................................................... 52 
14.2 
Recruitment of Subjects ................................................................................................... 52 
14.3 
Study location and Performance Sites ............................................................................. 52 


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14.4 
Confidentiality .................................................................................................................. 52 
14.5 
Financial Obligations and Compensation ......................................................................... 52 
14.6 
Informed Consent Processes............................................................................................ 53 
15.0 
References ...................................................................................................................................... 54 
APPENDIX A: ECOG Performance Status Criteria .......................................................................................... 57 
APPENDIX B. Detailed Methods for Pharmacogenomic, Ex Vivo, and Molecular Studies ............................ 58 
 


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ABBREVIATIONS 
Abbreviation 
Meaning 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
COH 
City of Hope 
CR 
Complete Response 
CRC 
Clinical Research Coordinator  
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DLCO 
Carbon Monoxide Diffusing Capacity 
DLT 
DHODH 
Dose Limiting Toxicity 
Dihydroorotate Dehydrogenase  
DSMC 
EBMT 
Data Safety Monitoring Committee 
European Bone Marrow Transplant 
EOT 
End of Treatment 
FDA 
Food and Drug Administration 
FVC 
Forced Vital Capacity 
GCP 
Good Clinical Practice 
IB 
Investigator Brochure 
ICF 
Informed Consent Form 
ICG 
Integrated Genomics Core 
IDS 
IMWG 
Investigational Drug Services 
International Myeloma Working Group 
IND 
Investigational New Drug  
IRB 
MM 
MNC 
MR 
Institutional Review Board 
Multiple Myeloma 
Mononuclear Cells 
Minimal Response 
MTD 
Maximum Tolerated Dose 
NCI 
National Cancer Institute 
PD 
Progressive Disease 
PI 
Principal Investigator 
PMT 
Protocol Monitoring Team 
PR 
RP2D 
RRMM 
RA 
Partial Response 
Recommended Phase II Dose 
Relapsed Refractory Multiple Myeloma 
Rheumatoid Arthritis 
SAE 
Serious Adverse Event 
sCR 
SD 
Stringent Complete Response 
Stable Disease 
 
 


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1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
In patients with relapsed or relapsed/refractory multiple myeloma (MM): 
Phase I 
Primary Objectives: 
o 
To determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of 
leflunomide, when given as a single agent. 
o 
To assess the safety and tolerability of leflunomide at each dose level by evaluation of toxicities 
including: type, frequency, severity, attribution, time course and duration. 
 
Phase II 
Primary Objective: 
o To evaluate the anti-myeloma activity of leflunomide, when given as a single agent, as assessed 
by overall response rate (ORR). 
Secondary Objectives: 
o 
To obtain estimates of:  
o 
Response duration 
o 
Clinical benefit response 
o 
Overall survival and  
o 
Progression-free survival. 
 
Clinical Pharmacology Objectives (Phase I and II) 
o 
To characterize the relationship between serum concentration of the active leflunomide metabolite, 
teriflunomide (A77 1726), and toxicity. 
o 
To assess the relationship between serum concentration of the active leflunomide metabolite, 
teriflunomide A77 1726), and disease response.  
Exploratory Ex-Vivo and Molecular Objectives (Phase I and II) 
o 
To explore the relationship between polymorphisms in the CYP1A2, CYP2C19, or DHODH genes and 
toxicity/response. CYP1A2, CYP2C19 are the hepatic enzymes responsible for leflunomide 
metabolism. 
o To explore the ex vivo cytotoxicity of leflunomide toward primary MM cells, in order to evaluate 
whether individual ex vivo leflunomide response might be a useful predictor of therapeutic 
response.  
o To explore the potential additive or synergistic effects of combining leflunomide with other 
classes of FDA-approved drugs. The classes include HDAC inhibitors, immunomodulatory 
drugs [IMiDs], proteasome inhibitors, steroid hormones, and antibody therapeutics such as the 
CD38 antibody daratumumab. 
o To generate a preliminary RNA/miRNA and DNA methylation signature associated with response 
of MM cells to leflunomide in vivo (mRNA/miRNA and DNA methylation, phase II only) and 
teriflunomide ex vivo (mRNA/miRNA). 


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2.0 
BACKGROUND 
2.1 
Introduction/Rationale for Development 
Multiple myeloma (MM) is a plasma cell malignancy characterized by periods of remissions and relapses. 
Worldwide, the incidence of MM is about 86,000 cases annually with about 63,000 deaths each year [4]. 
In the United States, over 30,000 patients are expected to be diagnosed with MM in 2016 and more 
than 12,000 patients will die from the disease [5, 6].  
Treatments for MM include alkylating agents, immunomodulatory (IMiD) drugs, proteasome inhibitors, 
anthracyclines, and glucocorticoids. Therapeutic agents are used sequentially or in combination either 
as double, triple or quadruple chemotherapy combinations. While the prognosis for MM patients has 
improved [7, 8], MM remains an incurable disease with a median survival of approximately 3 to 4 years 
with standard therapy[9]. The 5-year survival rate for patients with MM is estimated at 46% (Cancer 
Statistics 2016), with only 17% of patients surviving for 10 years[10].  
Relapsed and refractory multiple myeloma (RRMM) is defined as disease that is nonresponsive while on 
salvage therapy, or progressing within 60 days of cessation of the most recent prior therapy in patients 
who have achieved minimal response (MR) or better[11]. Primary refractory myeloma is defined as 
disease that is nonresponsive in patients who have never achieved a minimal response or better with 
any therapy[11]. The overall survival for patients who have received more than 3 prior regimens and 
whose disease has relapsed after treatment with bortezomib and lenalidomide is estimated to be 
between 6 and 10 months [12, 13]. Treatment decision making in the RRMM population is particularly 
challenging, given underlying comorbidities and the toxic nature of available agents. Moreover, duration 
of response to therapies is traditionally short due to multi-drug resistance as well as changes in disease 
biology (e.g., development of aggressive phenotype, higher proliferation rates, and lower apoptotic 
rates) [14]. Patients with RRMM are in dire need of new treatments with new mechanisms of action that 
could be used successfully long term. 
2.2 
Leflunomide 
Leflunomide is a commercially available oral immunosuppressive agent that has been FDA approved 
since 1998 for the treatment of rheumatoid arthritis (RA) as a single agent or in combination with 
methotrexate. It has been used in over 300,000 patients worldwide for RA treatment. Leflunomide is 
generally well-tolerated and may be taken over a long period of time.  
The in vitro and in vivo mechanisms of leflunomide are not completely defined. The primary clinical 
mechanism of action is inhibiting de novo pyrimidine synthesis by targeting dihydroorotate 
dehydrogenase (DHODH), and thus achieving anti-proliferative effect in B- and T-lymphocytes. A 
secondary mechanism of action is inhibition of cytokine and growth factor receptor associated tyrosine 
kinase activity [15].  
Leflunomide (the pro-drug) is rapidly converted to its active primary metabolite teriflunomide (A77 
1726), which mediates leflunomide’s pharmacologic activity. Teriflunomide serum concentrations show 
wide inter-patient variability; in one study, the mean serum teriflunomide concentration in patients 
receiving 20 mg/ daily was 42 mg/l with a range from 3-150 mg/l and a standard deviation of 35 mg/l 
[16]. Of note, the serum concentration of this metabolite was shown to predict RA response[16].  
The FDA approved dose in adults for the treatment of RA is a loading dose of 100 mg orally (PO) once 
daily for 3 days, followed by a maintenance dose of 10-20 mg PO once daily. Leflunomide has been used 
at up to 40 mg/day in patients with Wegner’s granulomatosis with a safety profile similar to the 
20mg/day dose used in RA [17, 18]. Doses up to 60 mg/day have been safely used for the treatment of 
allograft polyoma BK virus in renal allograft patients to achieve a targeted blood level of teriflunomide 


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(A77 1726) of 50-100 micrograms/mL [19]; the targeted blood level was frequently achieved with a dose 
of 40mg/ day with no increase in toxicity reported [19].  
In addition to studies that demonstrate the serum concentration of the metabolite may predict 
response, recent work demonstrates that polymorphisms in the gene encoding DHODH may be 
associated with leflunomide treatment outcome in RA patients[20, 21].  
The long half-life of the metabolite (about 2 weeks) affects management of patients taking leflunomide. 
A loading dose of 100 mg for 3 days is used to facilitate rapid attainment of steady state levels of M1. 
Without a loading dose, it is estimated that attainment of steady state concentrations would require 
nearly two months of dosing [22]. To eliminate the metabolite, a drug elimination procedure using 
cholestyramine is recommended. Cholestyramine, a bile acid sequestrant, fixes the metabolite, 
preventing reabsorption and expediting drug elimination. Without the drug elimination procedure, it 
may take up to 2 years to reach non-detectable plasma levels after stopping treatment with leflunomide 
[22].  
2.3 
Preclinical Studies of Leflunomide as an Anti-Neoplastic Agent 
Leflunomide has been investigated for its anti-neoplastic potential in a variety of pre-clinical tumor 
models. The first reports of anti-tumor activity in mouse xenograft models of glioma indicate that 
inhibition of pyrimidine synthesis is not sufficient for anti-tumor activity and concomitant inhibition of 
tyrosine phosphorylation may also play a role[23]. In chronic lymphocytic leukemia (CLL), leflunomide 
has been shown to inhibit the cell cycle progression in primary leukemic cells in vitro [24]. Dietrich et al 
showed that teriflunomide (A77 1726) affects proliferation of CLL by blocking DHODH at very low 
concentration (3-5 µg/ml) and by additionally inhibiting JAL/STAT pathway at intermediate 
concentrations (>10 µg/ml)[25]. The metabolite is also effective in fludarabine-refractory cells[25]. In 
addition, pre-clinical anti-tumor activity of leflunomide has been reported for melanoma [26] and 
prostate cancer [27].  
Recent pre-clinical studies of teriflunomide (A77 1726) have demonstrated significant antineoplastic 
activity in myeloma [28]. Teriflunomide inhibits cell growth and induces apoptosis in common myeloma 
cell lines at clinically achievable concentrations (50-200 µmol/L) in a time- and dose-dependent manner. 
The stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on multiple myeloma 
cell growth is completely abrogated by teriflunomide. In addition, studies revealed additive effects when 
teriflunomide was combined with the genotoxic agents melphalan and doxorubicin and synergistic 
effects when combined with bortezomib and treosulfan[28]. Finally, the Rosen laboratory has found 
leflunomide to be active in both glucocorticoid sensitive and resistant myeloma cell lines (Steven Rosen, 
personal communication).  
2.4 
Clinical Experience with Leflunomide for Multiple Myeloma 
Oral leflunomide was given to an 81-year-old female myeloma patient who did not have other 
treatment options or suitable clinical trials. This patient was diagnosed with myeloma in January of 2001 
and had an extensive treatment history including Velcade, Velcade-Doxil, Revlimid-dexamethasone, 
Revlimid-Melphalan-Prednisone, and Velcade-dexamethasone. Her disease was refractory and 
progressive, with rapidly increasing IgG, M protein, and pancytopenia with transfusion-dependent 
anemia. She began oral daily leflunomide (20 mg per day) in February 2011. Her myeloma showed a 
gradual and significant improvement, with reduction of her serum IgG (from 6280 to 3890 mg/dl), M-
protein, and beta-2-microglobulin levels. Her anemia improved so that she was no longer transfusion-
dependent. She tolerated the treatment fairly well except for mild diarrhea managed with loperamide. 
She remained on leflunomide for 16 months with continued clinical response of her myeloma but 
unfortunately died of her other medical problems (severe cardiac valvular disease and congestive heart 
failure).  


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2.5 
Background for Correlative Studies 
Leflunomide is rapidly converted to its active primary metabolite teriflunomide (A77 1726), which 
mediates leflunomide’s pharmacologic activity. Previous population-based PK studies in patients with RA 
have demonstrated a high degree of intersubject variability in measured steady-state total (free + 
albumin-bound) teriflunomide serum concentrations, resulting in coefficients of variation in CL/F 
estimates of >50% [29, 30]. Teriflunomide is >99% bound to serum albumin and free drug concentrations 
have also been reported to be highly variable, with a median of 55.8 μg/l and a range of 27.9-148.4 μg/l 
[31]. Moreover, measured total and free teriflunomide levels have been shown to predict response and 
toxicity in RA patients, leading some investigators to suggest a role for therapeutic drug monitoring and 
dose optimization of leflunomide [16]. It has also been reported that genetic polymorphisms in CYP1A2 
and CYP2C19, the hepatic enzymes responsible for leflunomide metabolism, and DHODH, a key enzyme 
in the de novo synthesis of pyrimidines and the main target of teriflunomide, can all influence the risk of 
leflunomide-associated toxicity in RA patients [32-34]. DHODH is expressed in MM cells, however, the 
relationship between DHODH gene polymorphism and toxicity/response to leflunomide is not well 
understood. Therefore, a better understanding of the relationship between teriflunomide serum 
concentrations and toxicity/response, as well as the influence of inherited polymorphisms in the genes 
involved in the metabolism and mechanism of action of leflunomide is needed. 
Various distinct genomic and epigenomic abnormalities have been observed in multiple myeloma, 
including gene mutations, chromosomal abnormalities, and DNA methylation [35]. Inhibition of tumor 
suppressor gene expression by DNA hypermethylation promotes survival and expansion of MM cells and 
clinical outcomes have been recently linked to DNA methylation status of MM cells [36]. Molecular 
signatures capturing such gene expression data have also recently emerged as powerful tools to identify 
patients who might respond particularly well to a specific treatment. Although genomic and epigenomic 
markers show promise for MM, there are still very few studies available and reliable markers have not 
yet emerged.  
2.6 
Overview and Rationale of Proposed Study 
The pre-clinical data, along with limited although promising clinical data of leflunomide for treatment 
MM, and the relatively low toxicity profile seen with prolonged administration in the rheumatoid 
arthritis population, in total provide evidence to support the investigation of leflunomide as an anti-
myeloma agent. This single agent phase I/II dose-escalation trial is designed to determine the MTD and 
to generate information about efficacy at theRP2D. Relapsed or relapsed/refractory multiple myeloma 
(RRMM) patients with documented measurable disease after at least three prior regimens will be 
considered for enrollment.  
While the standard dose of leflunomide (20mg daily), following a loading dose of 100mg daily for 3 days, 
is FDA approved for continuous treatment of rheumatoid arthritis, leflunomide has been administered 
without an increase in the type, frequency or severity of adverse events at doses up to 40 mg/day in 
patients with Wegner’s granulomatosis, and up to 60mg/day in a subset of patients with polyoma BK 
neuropathy to achieve targeted therapeutic levels [17, 18].  
Because the current dosing guideline for patients with rheumatoid arthritis do not to exceed 20 mg daily 
maintenance doses [22], the 20mg/day dose is selected as the safe starting dose. We will attempt to 
escalate the dose of leflunomide to three times that approved for RA using a modified rolling six dose-
escalation/ de-escalation/expansion design, a more conservative version of the rolling six design of 
Skolnik, et al.[1]. The starting dose of leflunomide is 20mg daily, with the intention to test up to 60mg 
daily. Escalation will proceed in increments of 20mg/day (odd numbered dose levels); de-escalation will 
proceed in decrements of 10mg/day.  


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Patients will be treated in planned 28-day treatment cycles. Patients may continue to receive cycles of 
treatment on study until disease progression or unacceptable toxicity, or other criteria for removal from 
study treatment are satisfied. While on active treatment patients will undergo monthly evaluation for 
response using the International Myeloma Working Group (IMWG) criteria [37, 38]. Following the 
decision to end leflunomide treatment, all patients will undergo 11 days of treatment with 
cholestyramine, which fixes the metabolite, preventing re-absorption and effectively lowering drug 
levels. Cholestyramine has been shown to decrease plasma levels of teriflunomide (A77 1726) in healthy 
volunteers by approximately 40% in 24 hours and by 49-65% in 48 hours [22]. 
Previous RA studies have shown a large variation in the serum concentration of the primary active 
metabolite, teriflunomide (A77 1726), and a suggested association between serum concentration of this 
metabolite and response [19].  Although the prior PK/PD relationship was identified in a non-oncologic 
patient population, it is possible that a similar association could exist for leflunomide exposures and 
antitumor response. Therefore, as part of the secondary objectives for this trial, enrolled RRMM 
patients will have blood samples collected and then processed by the Analytical Pharmacology Core 
Facility (APCF) to assess the possible relationship between serum concentration of the active 
leflunomide metabolite (teriflunomide) and toxicity/response. The natural PK variability of leflunomide 
administered at fixed doses will allow us to assess patients over a wide range of measured drug 
exposures. If a therapeutic window for leflunomide exposure can be identified, future studies based on 
targeted drug exposures will be considered. Additional testing will be done on collected blood samples 
to assess, in an exploratory manner, the potential association between germline polymorphisms in the 
CYP1A2, CYP2C19, and DHODH gene and either toxicity or response.  
In addition to the PK/PD analysis, additional molecular correlates and ex vivo responses will be 
evaluated using bone marrow specimens, which will be collected from patients before treatment and 
after one month of leflunomide treatment. MM cells will be purified from the pre-treatment specimens 
and treated with teriflunomide to determine the ex vivo IC50. The goal of this study is to determine 
whether ex vivo drug response predicts in vivo therapeutic response. The ex vivo treatment 
concentrations will be similar to in vivo drug levels. Teriflunomide will also be combined with drugs from 
other classes (e.g., HDAC inhibitors, immunomodulatory drugs [IMiDs], proteasome inhibitors, steroid 
hormones, and antibody therapeutics such as the CD38 antibody daratumumab), in order to evaluate ex 
vivo synergy and potentially predict the success of combination therapies. Molecular correlate analysis 
of leflunomide response will include miRNA, mRNA and DNA methylation profiling. Specimens derived 
from before and after leflunomide treatment (after 1 month and at progression or complete response, if 
possible) will be analyzed, as well as pre-treatment specimens exposed to teriflunomide ex vivo. The 
miRNA, mRNA and DNA methylation profiling analysis of specimens derived from participants treated 
with leflunomide in vivo will be conducted only for participants treated at the RP2D in order to minimize 
extra interventions for participants who may not receive a therapeutically effective dose. 
Following completion of the phase I portion of the study and identification of the RP2D, the phase II 
portion of the study will be initiated. Recent studies evaluating daratumumab, a CD38 monoclonal 
antibody, as monotherapy for relapsed/refractory multiple myeloma were recently published [39, 40] 
and have led to FDA approval of this agent. One study included patients with relapsed multiple myeloma 
treated with at least three lines of therapy while the other included patients treated with at least two 
prior lines of treatment. Treatment with daratumumab as a single agent lead to an overall response rate 
of 29.2% and 36% respectively in each of these studies. In both studies, the agent investigated was well 
tolerated but required IV dosing once weekly for 8 doses, followed by twice-monthly doses for 8 doses 
and then monthly thereafter. We suspect leflunomide to be similarly well tolerated but with a more 
convenient dosing schedule. Because we are including patients who have received at least three prior 
lines of treatment, we will target an overall response rate of 30% for the phase II portion of the study.   
 
 


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3.0 
PATIENT ELIGIBILITY 
3.1 
Inclusion Criteria  
Participants must meet the following criteria on screening examination to be eligible to participate in 
the study: 
Patient MRN: 
Patient Initials (F, M, L): 
Informed Consent 
___ 1. 
All subjects must have the ability to understand and the willingness to sign a 
written informed consent. 
Age Criteria, Performance Status, and Life Expectancy 
___ 2. 
Patients must be age ≥ 18 years. 
___ 3. 
Patients must have a life expectancy of > 3 months. 
___ 4. 
Patients must exhibit an ECOG performance status of 0-2. 
Nature of Illness and Treatment History 
___ 5. 
Patients must have a diagnosis of multiple myeloma. 
___ 6. 
Patients must have measureable disease, defined as one of the following 
within 21 days prior to registration: 
o 
Serum M-protein ≥ 0.5 g/dL 
o Urine M-protein ≥ 200 mg/24 hr 
o Serum free light chain ≥ 10 mg/dL provided the FLC ratio is abnormal. 
o 10% plasma cells in bone marrow 
___ 7. 
Patients must be relapsed or are refractory to at least 3 prior lines of therapy, 
including both a proteasome inhibitor and an IMiD, and for whom transplant is 
not recommended (induction therapy and stem cell transplant +/- maintenance will 
be considered as one regimen). 
___ 8. 
At least 2 weeks from prior therapy to time of start of treatment. Prior 
therapy includes steroids (except prednisone or equivalent – up to 10 mg per day 
is allowed). 
Clinical laboratory parameters  
___ 9. 
Platelet count > 50,000/µL. Platelet transfusions are not allowed within 14 
days of platelet assessment.  
___ 10. ANC ≥ 1000/mm3. Growth factor is not permitted within 14 days of neutrophil 
assessment. 
___ 11. AST and ALT < 2.0 x ULN. 
___ 12. Total Bilirubin < 1.5 x ULN. 
___ 13. Calculated creatinine clearance (CrCl) ≥ 30 mL/min per 24-hour urine 
collection or the Cockcroft-Gault formula. 


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CrCl (mL/min) = 
(140-age) x actual body weight (kg) 
( x 0.85 for females) 
72 x serum creatinine (mg/dL) 
 
 
 
 
Or 
CrCl (mL/min) = 
(140-age) x actual body weight (kg) 
( x 0.85 for females) 
0.8136 x serum creatinine (umol/L) 
___ 14. Negative serum or urine β-HCG test (female patient of childbearing potential* 
only), to be performed locally within the screening period.  
___ 15. Negative for tuberculosis antigen (e.g. T-Spot test). 
___ 16. Negative for hepatitis A, B, or C infection.  
___ 17. Adequate pulmonary function as defined by FVC and DLCO ≥ 50% of predicted 
by pulmonary function testing.   
Child Bearing Potential 
___ 18. Agreement by females of childbearing potential* and sexually active males to 
use an effective method of contraception (hormonal or barrier method of birth 
control or abstinence) prior to study entry and for three months following 
duration of study participation. The effects of study treatment on a developing 
fetus have the potential for teratogenic or abortifacient effects. Should a woman 
become pregnant or suspect that she is pregnant while participating on the trial, 
she should inform her treating physician immediately.  
*A female of childbearing potential is defined as a sexually mature woman who: 1) has not undergone a 
hysterectomy or bilateral oophorectomy; or 2) has not been naturally postmenopausal for at least 24 
consecutive months. 
3.2 
Exclusion Criteria  
Prospective participants who meet any of the following criteria will not be eligible for admission into the 
study: 
Previous therapies 
___ 19. Prior treatment with leflunomide. 
Concomitant medications 
___ 20. Current or planned use of other investigational agents, or concurrent 
biological, chemotherapy, or radiation therapy during the study treatment period. 
(See inclusion criterion #8 for washout times). 
___ 21. Current or planned growth factor or transfusion support until after initiation 
of treatment. If growth factor or transfusion support is provided between 
screening and start of treatment, the participant will no longer be eligible.  
Other illnesses or conditions 
___ 22. Prior diagnosis of rheumatoid arthritis. 
___ 23. Prior Allogeneic transplant. 
___ 24. Acute active infection requiring systemic therapy within 2 weeks prior to 
enrollment. 


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___ 25. Pre-existing liver disease.  
___ 26. Known HIV infection. 
___ 27. History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to leflunomide or cholestyramine. 
___ 28. Non-hematologic malignancy within the past 3 years aside from the following 
exceptions:  
o adequately treated basal cell or squamous cell skin cancer 
o carcinoma in situ of the cervix 
o prostate cancer < Gleason Grade 6 with a stable PSA 
o successfully treated in situ carcinoma of the breast 
___ 29. Clinically significant medical disease or condition that, in the investigator’s 
opinion, may interfere with protocol adherence or the patient’s ability to give 
informed consent.  
___ 30. Pregnant women and women who are lactating. Leflunomide has potential for 
teratogenic or abortifacient effects. Because there is a potential risk for adverse 
events in nursing infants secondary to treatment of the mother with these agents, 
breastfeeding should be discontinued if the mother is enrolled on this study.  
___ 31. Any other condition that would, in the Investigator’s judgment, contraindicate 
the patient’s participation in the clinical study due to safety concerns or 
compliance with clinical study procedures, e.g., infection/inflammation, intestinal 
obstruction, unable to swallow medication, social/ psychological issues, etc. 
Noncompliance 
___ 32. Prospective participants who, in the opinion of the investigator, may not be 
able to comply with all study procedures (including compliance issues related to 
feasibility/logistics). 
3.3 
Inclusion of Women and Minorities 
The study is open anyone regardless of gender or race/ethnicity. Efforts will be made to extend the 
accrual to a representative population. However, a balance must be struck between subject safety 
considerations and limitations on the number of individuals exposed to potentially toxic or ineffective 
treatments on the one hand and the need to explore gender, racial, and ethnic aspects of clinical 
research on the other. If differences in outcome that correlate to gender, racial, or ethnic identity are 
noted, accrual may be expanded or additional studies may be performed to investigate those 
differences more fully. 
4.0 
SCREENING AND REGISTRATION PROCEDURES 
4.1 
Screening Procedures 
Diagnostic or laboratory studies performed exclusively to determine eligibility for this trial will be done 
only after obtaining written informed consent. Studies or procedures that were for clinical indications 
(not exclusively to determine study eligibility) may be used for baseline values, even if the studies were 
done before informed consent was obtained. All screening procedures and their respective windows are 
detailed in Section 10, Table 10, Study Activity Calendar. 


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4.2 
Informed Consent  
The investigational nature and objectives of the trial, the procedures and treatments involved and their 
attendant risks and discomforts, and potential alternative therapies will be carefully explained to the 
subject and a signed informed consent will be obtained. Documentation of informed consent for 
screening will be maintained in the subject’s research chart and medical record and the patient will 
receive a copy of the signed informed consent document. All Institutional, Federal, and State regulations 
concerning Informed Consent will be fulfilled. 
4.3 
Registration Requirements/Process 
Confirming, reserving a slot and dose level assignment 
Eligible subjects will be registered on the study centrally by the Data Coordinating Center (DCC) at City of 
Hope. Staff (including physicians, protocol nurses and/or CRCs) should call the DCC at (626) 256-4673, 
ext. 63968 to verify the current dose level and slot availability, and to reserve a slot for a specific 
prospective subject. Slots can only be held for a limited time.  
Eligible subjects must be registered prior to start of protocol therapy. Issues that would cause treatment 
delays should be discussed with the Principal Investigator. If a subject does not receive protocol therapy 
following registration, the subject’s registration on the study may be canceled after discussion with the 
PI. The Data Coordinating Center should be notified of cancellations as soon as possible. 
Registration Process  
Once a slot at a dose level has been reserved, the signed informed consent has been obtained, all 
pretreatment evaluations have been performed, and subject’s eligibility has been confirmed by the Data 
Coordinating Center, a subject will be registered on study. 
To register a subject, the treating physician should contact the protocol nurse or the responsible Clinical 
Research Coordinator (CRC) in the Clinical Trial Office (CTO) to complete the eligibility checklist.  
The protocol nurse or CRC will contact the Data Coordinating Center at City of Hope (626-256-4673, ext. 
63968 or via e-mail at dcc@coh.org), scan and EMAIL a copy of the completed and signed eligibility 
checklist, a copy of the signed Informed Consent (including a copy of signed subject’s bill of Rights and 
HIPAA authorization form) and copies of any required pre-study test results that are not readily available 
in the COH electronic medical record to dcc@coh.org.  
The protocol nurse or CRC may then call the Data Coordinating Center (626-256-4673 ext. 63968) to 
confirm receipt of all registration documents. To complete the registration process, the Data 
Coordinating Center will: 
 
Verify and confirm the subject’s eligibility. 
 
Assign a subject accession number (for example, COH-001, COH-002, etc.). 
 
Register the subject on study centrally (the City of Hope CRC assigned to the trial will still be 
responsible for accessioning via MIDAS).  
 
Assign the subject to a dose level (as applicable). 
 
Complete and email a Confirmation of Registration form within 24 hours to include the COH 
subject study number and dose level to the study team, which will include the Principal 
Investigator, treating physician, protocol nurse, CRC and COH IDS Pharmacy. 
 
Call the protocol nurse and/or CRC to verbally confirm registration. 
 
Enter the subject into Medidata RAVE®. 
 
A subject failing to meet all protocol requirements will not be registered. 


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Patients must begin protocol treatment within 1 week following registration date.  
4.4 
Screen Failures and Registered Participants Who Do Not begin Study Treatment 
The reason for screen failure or failure to begin treatment for registered participants will be 
documented.  
5.0 
TREATMENT PROGRAM  
5.1 
Treatment Overview 
Treatment on study will be in the outpatient setting and will consist of daily treatment with leflunomide; 
patients will be treated in planned 28-day treatment cycles. After the decision to end study treatment 
has been made, participants will receive treatment with cholestyramine (Section 5.7) to eliminate 
leflunomide. Participants who end study treatment for reasons other than disease progression will 
undergo active follow-up (Section 5.9) until disease progression or the initiation of a new therapy. All 
participants will be followed for survival (Section 5.9).  
For a detailed tabular view of the treatment, monitoring, and follow-up schedule, see the Study 
Calendar in Section 10.  
5.2 
Treatment cycle definition 
The planned cycle length is 28 days. Leflunomide will be taken orally once daily. Missed or held doses 
will not be made up. The day count continues despite a hold in agent administration. Day 1 safety 
assessments must be reviewed prior to the initiation of the cycle. For a cycle to commence, the 
participant must be actively taking study agent. Windows for procedures are detailed in Section 10. 
5.3 
Phase I/II Treatment Plan 
This phase I trial will implement a modified rolling six dose escalation, de-escalation, expansion design, a 
more conservative version of the rolling six design of Skolnik, et al. [1] to ensure safety and to allow for 
the evaluation of toxicities associated with leflunomide. See Section 13.3.2 for dose escalation/de-
escalation/expansion rules, and Section 13.1 for the definition of dose limiting toxicity (DLT). 
Up to six doses of leflunomide will be considered (see Table 5.3.1). The starting dose of leflunomide is 
20mg daily, with the intention to test up to 60mg daily. Escalation will proceed in increments of 
20mg/day (odd numbered dose levels); de-escalation will proceed in decrements of 10mg/day. There 
will be no intra-patient dose escalation; participants will undergo dose modification due to toxicity per 
Section 6.2.  


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Table 5.3.1: Dose Levels  
Dose Level 
Leflunomide Dose* 
-1 
10 mg PO daily 
1 
20 mg PO daily 
2 
30 mg PO daily 
3 
40 mg PO daily 
4 
50 mg PO daily 
5 
60 mg PO daily 
*NOTE: Leflunomide is administered with a loading dose of 100 mg for the 1st three doses. 
5.4 
Phase II Treatment Plan 
Following the phase I trial, a single arm phase II study will be conducted using a Simon Two-Stage 
Optimal Design [2, 3] to evaluate the anti-myeloma activity of leflunomide. For the phase II portion, up 
to 39 patients will be treated. The six patients treated at the RP2D in the phase I portion of the study will 
count toward the 39 patients required; given this, we expect to enroll only 33 new patients on the phase 
II trial.   
5.5 
Agents Administration 
5.5.1 
Leflunomide 
Participants will be instructed to take each dose of leflunomide orally, once a day, at approximately the 
same time each day. They may take it with or without food. If a patient forgets or misses a dose for 
whatever reason, it should not be replaced or made up. Participants will be given a study calendar (pill 
diary) to document each dose of leflunomide that is taken or missed.  
NOTE: On study visit days, participants should refrain from taking leflunomide until after blood is 
collected.  
Leflunomide is administered with a loading dose of 100 mg per day for the first three doses (first three 
days). 
There will be no intra-patient dose escalation; patients will undergo dose modification due to toxicity 
per Section 6.2.  
5.5.2 
Cholestyramine 
Cholestyramine is not a study agent but is required for the elimination of the study agent leflunomide. 
See Section 5.7 for cholestyramine administration details. 
5.6 
Study Procedures 
For a detailed list of all study procedures including timing and windows, see Section 10.  
5.7 
End of Treatment Evaluations and Leflunomide Detoxification 
All participants will be followed until resolution or stabilization of any serious adverse events occurring 
during treatment and completion of the Day 30 End of Treatment assessments. See Section 10 for a list 
of all assessments and windows. 


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Because of the ability of leflunomide to be reabsorbed through the colon, resulting in a very long half-
life, all participants will be treated with cholestyramine to prevent re-absorption of leflunomide after 
the decision to end study treatment has been made.  
Participants removed from study treatment will undergo oral administration of cholestyramine 8 grams 
oral suspension three times daily for 11 days. It will not be a deviation if an administration of 
cholestyramine is delayed or if the 11 days are not consecutive. Cholestyramine administration may be 
adjusted per the discretion of the treating investigator. After the treatment with cholestyramine, clinical 
laboratory testing will be performed to verify plasma levels less than 0.02 mg/L or 0.02µg/mL by two 
separate tests at least 14 days apart. If plasma levels are higher than 0.02 mg/L or 0.02µg/mL, additional 
cholestyramine treatment will be considered. See Section 10 for a tabular view of cholestyramine 
administration and metabolite testing windows and days of administration. 
Note: Cholestyramine can affect the absorption of other oral medications. Such medications should be 
taken 1 hour before the first administration or per other guidance in the package insert. Watch for 
bleeding abnormalities due to vitamin K deficiency for which intravenous vitamin K may be 
administered.  
5.8 
Duration of Therapy & Criteria for Removal from Study Treatment 
Patients will receive study treatment until disease progression, unacceptable toxicity or other criteria for 
removal from study treatment are satisfied. Of note, based on the guidelines outlined by Rajkumar et al. 
[38], “CR patients will need to progress to the same level as VGPR and PR patients to be considered PD. 
A positive immunofixation alone is therefore not sufficient.” Participants may be removed from 
treatment for any of the following reasons: 
o Evidence of disease progression 
o Patient is deemed intolerant to study treatment because of toxicity, despite dose 
modification/delay 
o Intercurrent illness that prevents further administration of treatment 
o Participant withdraws from the treatment phase of the study  
o General or specific changes in the participant's condition, including non-compliance, 
which renders the participant unacceptable for further treatment in the opinion of the 
treating investigator. 
Documentation of the reason for discontinuing therapy and the date effective should be made in the 
medical record and appropriate eCRF. The participant should then proceed to off-treatment procedures 
for safety monitoring, leflunomide elimination and follow-up procedures. The participant’s status is to 
be modified in the MIDAS system once the off-treatment period is completed. Alternative care options 
will be discussed with the participant. 
5.9 
Active Follow Up and Long Term Follow Up 
Participants who do not progress while on treatment will be in Active Follow Up, where they will 
continue to undergo monthly response assessment (myeloma labs) until disease progression or the 
initiation of a new therapy. 
Participants who progress during Treatment Phase skip Active Follow-Up phase and continue directly 
into Long-term Follow-up phase.  
In Long-term Follow-Up participants will be followed for survival information which may be obtained by 
reviewing the City of Hope medical record, contacting the participant and/or a review of outside medical 
records. Note: this is not intended to be an all-inclusive list. 
The schedule and windows for assessments and data collection points are further detailed in Table 10. 


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5.10 Criteria for Completion of Study Participation 
Participants may be removed from the study as a whole at any time at their own request, or they may 
be withdrawn at the discretion of the investigator for safety, behavioral or administrative reasons. The 
reason(s) for discontinuing study participation should be documented and may include: 
o Participant completes all study procedures including all study follow-up procedures. 
o Participant withdraws consent for follow-up. 
o Participant is determined to be lost to follow-up. All attempts to contact the subject 
must be documented. 
The reason for study removal and the date the participant was removed must be documented in 
the source documentation and the study-specific case report form (CRF). The participant’s status 
is to be modified in the MIDAS system once the participant completes the study.  
5.11 Supportive Care, Other Concomitant Therapy, Prohibited Medications 
Concomitant medications and treatments that are permitted while on study treatment include 
allopurinol, anti-emetics, anti-diarrheals, FDA-approved bisphosphonates, erythropoietin, transfusions 
(as necessary), and palliative radiation. 
Concurrent treatment with any other anti-neoplastic therapies will not be permitted while on study 
treatment. Concurrent administration of live vaccines will not be permitted while on study treatment. 
During cycle 1 only: Transfusions are not permitted unless platelets <25,000/mm3 or platelets < 50,000/ 
mm3 accompanied by bleeding. Growth factors are not permitted unless ANC <500/ mm3. 
5.12 Additional Studies 
See Section 9.0 for correlative studies. 
6.0 
ANTICIPATED TOXICITIES AND DOSE DELAYS/MODIFICATIONS FOR ADVERSE EVENTS 
6.1 
Anticipated Toxicities 
6.1.1 
Leflunomide 
Per the package insert for leflunomide, the expected toxicities for leflunomide follow, where the asterisk 
(*) signifies a common event (10%-30% of patients), unmarked items are infrequent events (1-3% of 
patients), a double asterisk (**) signifies a rare but possibly serious event, and a triple asterisk (***) 
signifies an event that may become serious and occurs in up to 10% of patients:  
Blood and lymphatic system: anemia (including iron deficiency anemia), ecchymosis, 
pancytopenia**, 
agranulocytosis**, 
neutropenia**, 
thrombocytopenia**, 
leukopenia**, 
Cardiac: angina pectoris, migraine, palpitation, tachycardia, vasodilatation 
Endocrine: diabetes mellitus, hyperthyroidism 
Eye: blurred vision, cataract, conjunctivitis, eye disorder 
Gastrointestinal: diarrhea*, abdominal pain, dyspepsia, nausea, vomiting, oral 
ulceration, anorexia, pancreatitis**, constipation, esophagitis, flatulence, gastritis, 
gingivitis, melena, oral moniliasis, pharyngitis, salivary gland enlarged, stomatitis (or 
aphthous stomatitis), tooth disorder 


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Hepatobillary: cirrhosis**, hepatitis**, hepatic failure**, acute hepatic necrosis**, 
cholelithiasis**, cholestasis**, elevated hepatic enzymes (primarily ALT and AST) 
Immune system: allergic reactions, anaphylactoid reactions** 
Infections: respiratory infection*, infections (including bronchitis, rhinitis, sinusitis, 
pharyngitis, pneumonia, and urinary tract infections, oral or vaginal candidiasis, herpes 
simplex, herpes zoster, and fungal dermatitis), opportunistic and/or severe infections** 
including sepsis, that may be fatal (especially Pneumocystis jiroveci pneumonia, 
tuberculosis, aspergillosis) 
Metabolism and nutrition: hyperglycemia, creatine phosphokinase increased, 
hyperlipidemia 
Musculoskeletal and connective tissue: arthrosis, bone necrosis, bone pain, bursitis, 
muscle cramps, myalgia, tendon rupture 
Neoplasms: secondary malignancy**, cyst 
Nervous system: headache*, peripheral neuropathy* (including peripheral numbness, 
tingling, burning, severe pain, cold sensation in the distal extremities, or extremity 
weakness), paresthesias, taste perversion (dysgeusia), anxiety, depression, dry mouth, 
insomnia, neuralgia, neuritis, sleep disorder, sweating increased, vertigo, migraine 
Renal and urinary: albuminuria, cystitis, dysuria, hematuria, hypophosphaturia, 
hyperuricemia, increased urinary frequency 
Reproductive: menstrual irregularity/disorder, menstrual disorder, vaginal moniliasis 
vaginal moniliasis, prostate disorder 
Respiratory: interstitial lung disease** (sometimes fatal), interstitial pneumonitis**, 
pulmonary fibrosis**, asthma, dyspnea, epistaxis, lung disorder 
Skin and subcutaneous tissue: maculopapular rash*, dry skin*, alopecia*, hair 
discoloration*, Stevens-Johnson syndrome**, toxic epidermal necrolysis**, erythema 
multiforme**, cutaneous lupus erythematosus**, acne, contact dermatitis, fungal 
dermatitis, hematoma, nail disorder, skin discoloration, skin disorder, skin nodule, 
subcutaneous nodule, skin ulcer 
Vascular: varicose veins, hypertension***, vasculitis, cutaneous necrotizing vasculitis** 
Miscellaneous: weight loss*, leg cramps*, jaundice**, allergy related angioedema**, 
fever, peripheral edema, hernia, neck pain, pelvic pain, pain, abscess, malaise 
6.1.2 
Cholestyramine 
Per the package insert for cholestyramine, the expected toxicities for cholestyramine follow, where the 
double asterisk (**) signifies a common event, and a single asterisk (*) signifies a less common event, 
and remaining unmarked items are less likely: 
Blood and lymphatic system: anemia, ecchymosis, prolonged prothrombin time, 
hypoprothrombinemia 
Ear and labyrinth: tinnitus, vertigo 
Eye: uveitis 
Gastrointestinal: constipation**, abdominal pain*, anorexia*, nausea*, vomiting*, 
diarrhea*, flatulence*, diarrhea*, eructation,* steatorrhea,* diverticulitis, bleeding from 
known duodenal ulcer, dysphagia, gastrointestinal hemorrhage, hemorrhoidal bleeding, 


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hiccups, intestinal obstruction (rare), melena, pancreatitis, rectal pain, tongue irritation, 
tooth enamel damage (dental erosion), dental bleeding, dental caries, dental 
discoloration 
Hepatobiliary: abnormal hepatic function tests, biliary colic, gallbladder calcification, 
Immunologic: hypersensitivity reaction 
Metabolism: hyperchloremic metabolic acidosis 
Musculoskeletal and connective tissue: arthralgia, arthritis, backache, myalgia, 
osteoporosis associated with vitamin D deficiency 
Nervous system: anxiety, dizziness, drowsiness, dysgeusia, fatigue, headache, neuralgia, 
paresthesia, syncope 
Respiratory: asthma dyspnea and wheezing associated with hypersensitivity reaction 
Renal and urinary: hematuria, dysuria, burnt odor to urine, diuresis 
Skin and subcutaneous tissue: perianal skin irritation, skin irritation, skin rash, urticaria  
Miscellaneous: adenopathy, increased libido, edema, bleeding tendencies due to 
vitamin K deficiency, vitamin deficiency (A, D, E, K), weight loss, weight gain, hematoma, 
hemorrhage, femoral nerve pain 
6.2 
Dose Modifications of Leflunomide 
6.2.1 
General Information 
a. The study will use the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.03 to grade toxicities. A copy of the version 4.03 can be downloaded from: 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html 
b. Intra-patient dose escalation is never permitted in this study. Rules for dose modification 
are found in Table 6.2.2. 
c. Baseline values are from the last values obtained prior to treatment.  
d. For holds due to toxicities related to study agent, if the participant does not meet criteria 
to resume treatment within 14 days, the participant must permanently discontinue study 
treatment.  
6.2.2 
Dose Modifications 
The tables below detail the specific dose modifications for toxicities on single agent leflunomide (Table 
6.2.2) and are to be used in agreement with the information in Section 6.2.1. 
Table 6.2.2 Dose Modifications for Leflunomide Treatment 
Adverse Event 
Treatment modification 
Hematological Toxicities:  
Febrile Neutropenia Grade 3 
(ANC: <1.0 x109/L with a single 
temperature of >38.3 ◦C or a 
sustained temperature ≥ 38.0◦C for 
more than one hour. 
Hold study agent. 
If resolves to ANC ≥ 1.0 x 109/L and temperature < 38.0◦C within 14 days, 
resume study agent at next lowest safe dose. 
If does not resolve within 14 days, permanently discontinue study agent. 
Febrile Neutropenia Grade 4 
Life threatening consequences; 
urgent intervention needed. 
Permanently discontinue study agent.  


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Neutropenia (ANC) Grade 1 
(1.5x 109/L -<LLN) 
Maintain study agent. 
Neutropenia (ANC) Grade 2 
(1.0 –<1.5x 109/L) 
Maintain study agent.  
Neutropenia (ANC) Grade 3 
(0.5 –< 1.0x 109/L) 
Maintain study agent. Growth factor recommended after cycle 1. 
Neutropenia (ANC) Grade 4 
(<0.5x 109/L) 
Hold study agent and provide supportive care. 
If ANC does not resolve to ≤ grade 3 (ANC ≥ 0.5 x 109/L) in 5 days, permanently 
discontinue study agent. Otherwise proceed as follows: 
 If resolves to ≤ grade 2 (ANC ≥ 1.0 x 109/L) in ≤ 14 days, resume study 
agent at the next lowest safe dose. 
 If ANC does not resolve to ≤ grade 2 (ANC ≥ 1.0 x 109/L) by 14 days, 
permanently discontinue study agent. 
Thrombocytopenia Grade 1 
(75 x 109/L -<LLN) 
Maintain study agent 
Thrombocytopenia Grade 2 
(50 – <75 x 109/L) 
Maintain study agent 
Thrombocytopenia Grade 3 without 
bleeding  
(25 – <50 x 109/L) 
Maintain study agent.  
Thrombocytopenia Grade 3 with 
bleeding 
(25 – <50 x 109/L) 
Hold study agent and provide supportive care. 
If bleeding does not resolve in 5 days, permanently discontinue study agent. 
Otherwise proceed as follows: 
 If bleeding resolves within 5 days and platelets resolve to ≤ grade 2 
(platelets ≥ 50 x 109/L) in ≤ 14 days, resume study agent at next lowest 
safe dose. If bleeding resolves within 5 days but thrombocytopenia 
does not resolve to ≤ grade 2 (platelets ≥ 50 x 109/L) by 14 days, 
permanently discontinue study agent. 
Thrombocytopenia Grade 4 
(<25 x 109/L) 
Hold study agent and provide supportive care. 
If platelets do not resolve to ≤ grade 3 (platelets ≥ 25 x 109/L) in 5 days, 
permanently discontinue study agent. Otherwise proceed as follows: 
 If resolves to ≤ grade 2 (platelets ≥ 50 x 109/L) in ≤ 14 days, resume 
study agent at next lowest safe dose. 
 If thrombocytopenia does not resolve to ≤ grade 2 (platelets ≥ 50 x 
109/L) by 14 days, permanently discontinue study agent. 
Gastrointestinal 
Diarrhea Grade 1 
(2-3 stools/day >baseline) 
Maintain treatment. 
Diarrhea Grade 2 
(4-6 stools/day > baseline) 
Maintain study agent. 
Diarrhea Grade 3 
(7-9 stools/day > baseline) 
 
Hold study agent and provide optimal anti-diarrheal therapy. 
First Occurrence:  
If resolves to ≤ grade 1 in ≤ 7 days, resume at pre-hold dose.  
If resolves to ≤ grade 1 in 8-14 days, resume study agent at next lowest safe 
dose.  
If does not resolve within 14 days, permanently discontinue study agent. 
Recurrence: 
If resolves to ≤ grade 1 in ≤ 14 days, resume at next lowest safe dose. 
If does not resolve within 14 days, permanently discontinue study agent. 


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Diarrhea Grade 4 
(≥10 stools/day > baseline) 
 
Hold study agent and provide optimal anti-diarrheal therapy.  
If does not resolve to Grade 3 within 48 hours, permanently discontinue study 
agent.  
If resolves to Grade 3 within 48 hours, then: 
First Occurrence:  
Hold until resolution to ≤ grade 1.  
If resolved in ≤ 7 days, resume at pre-hold dose.  
If resolved in 8-14 days, resume study agent at next lowest safe dose.  
If not resolved within 14 days, permanently discontinue study agent. 
Recurrence: 
Hold until resolution to ≤ grade 1. 
If resolved in ≤ 14 days, resume study agent at next lowest safe dose. 
     If not resolved within 14 days, permanently discontinue study agent. 
Vomiting or Nausea Grade 1 
Maintain study agent. 
Vomiting or Nausea Grade 2  
Maintain study agent. 
Vomiting or Nausea Grade 3 
Hold study agent and provide optimal supportive care/therapy. 
First Occurrence:  
If resolves to ≤ grade 1 in ≤ 7 days, resume at pre-hold dose.  
If resolves to ≤ grade 1 in 8-14 days, resume study agent at next lowest safe 
dose.  
If does not resolve within 14 days, permanently discontinue study agent. 
Recurrence: 
If resolves to ≤ grade 1 in ≤ 14 days, resume at next lowest safe dose. 
If does not resolve within 14 days, permanently discontinue study agent. 
Vomiting Grade 4 
Hold study agent and provide optimal supportive care/therapy.  
If does not resolve to Grade 3 within 48 hours, permanently discontinue study 
agent.  
If resolves to Grade 3 within 48 hours, then: 
First Occurrence:  
Hold until resolution to ≤ grade 1.  
If resolved in ≤ 7 days, resume at pre-hold dose.  
If resolved in 8-14 days, resume study agent at next lowest safe dose.  
If not resolved within 14 days, permanently discontinue study agent. 
Recurrence: 
Hold until resolution to ≤ grade 1. 
If resolved in ≤ 14 days, resume study agent at next lowest safe dose 
      If not resolved within 14 days, permanently discontinue study agent. 
Hepatic investigations 
ALT (SGPT) or AST (SGOT)  
< 3.0 x ULN or TBili < 1.5 x ULN 
Maintain study agent. 
ALT (SGPT) or AST (SGOT)   
>3.0 - 5.0 x ULN or TBili >1.5 - > 3.0 
x ULN 
 First Occurrence:  
Hold until resolution to < 3.0 x ULN for ALT or AST or < 1.5 for TBili.  Resume 
at pre-hold dose if resolved within 14 days. If does not resolve within 14 
days, permanently discontinue study agent. 
Recurrence: 
Hold until resolution to < 3.0 x ULN for ALT or AST or <1.5 for TBili. Resume 
at next lowest safe dose if resolved within 14 days. If does not resolve within 
14 days, permanently discontinue study agent. 
ALT (SGPT) or AST (SGOT)  
> 5.0 x ULN or TBili > 3.0 x ULN 
 
Permanently discontinue study agent. 
 
Electrolyte/metabolic toxicity 


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Electrolyte/metabolic Grade 3 
Provide support care. Maintain study agent per investigator discretion.  
 
If alteration persists despite aggressive replacement therapy, then dose reduce 
or permanently discontinue (per investigator discretion with PI consultation). 
Electrolyte/metabolic Grade 4 
Provide support care. Maintain study agent per investigator discretion. 
 
If resolves to Grade 1 or baseline within 48: for first event, maintain at pre-hold 
dose; for recurrent event, dose reduce or permanently discontinue (per 
investigator discretion with PI consultation). 
 
If does not resolve to Grade 1 or baseline within 48 hours, permanently 
discontinue study agent. 
Other unspecified Non-Hem toxicities considered related to leflunomide 
Grade 1 
Maintain study agent. 
Grade 2  
Maintain study agent. 
Grade 3 
First Occurrence:  
Hold until resolution to ≤ Grade 2. Resume at pre-hold dose if resolved 
within 7 days. If does not resolve within 7 days, permanently discontinue 
study agent. 
Recurrence: 
Hold until resolution to ≤ Grade 1. Resume at next lowest safe dose if 
resolved within 14 days. If does not resolve within 14 days, permanently 
discontinue study agent. 
Grade 4 
Permanently discontinue study agent. 
Other unspecified Non-Hem Toxicities considered UNRELATED to study agent 
Other unspecified events of any 
grade considered unlikely to be 
related or not related to study 
agents. 
Maintain treatment with study agents. Interruption of study agent or dose de-
escalation is permitted if the investigator consults with the Principal Investigator 
to determine that this is in the best interest of the participant.  
6.3 
Dose Modifications of Cholestyramine 
Cholestyramine will be administered and dose modified per the discretion of the treating investigator in 
agreement with standard administration practices.  
7.0 
DATA AND SAFETY MONITORING, UNANTICIPATED PROBLEM AND ADVERSE EVENT REPORTING  
7.1 
Definition of Risk Level 
This is a Risk Level 4 study as defined in the City of Hope Institutional Data and Safety Monitoring Plan 
[policy dated 07/09/2014]. This determination was made because the study involves COH as the IND 
holder and escalates the study agent to a dose above that indicated by the FDA for the agent’s currently 
approved indication. 
7.2 
Monitoring and Personnel Responsible for Monitoring 
The Protocol Management Team (PMT) consisting of the PI, Collaborating Investigator, CRC/protocol 
nurse, and statistician is responsible for monitoring the data and safety of this study, including 
implementation of the stopping rules for safety. 
The PMT is required to submit periodic status reports (i.e., the PMT Report) according to the frequency 
prescribed in the City of Hope Institutional Data and Safety Monitoring Plan [policy dated 07/09/2014]. 
Important decisions made during PMT meetings (i.e., dose escalation, de-escalation, etc.) only need to 
be noted in the PMT Report submitted to the Data and Safety Monitoring Committee (DSMC). 


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7.3 
Dose Escalation 
This study will utilize the Phase I Tracking Log to monitor data and safety for dose escalation. The 
Tracking Log will contain dose levels administered, dose limiting toxicities (DLT), DLT-defining adverse 
events, and any details regarding dose level escalation. The record of doses administered and resultant 
adverse events will be included in the PMT Report. 
7.4 
Adverse Events and Serious Adverse Events 
The PI will be responsible for determining the event name, assessing the severity (i.e., grade), 
expectedness, and attribution of all adverse events. 
Adverse Event (AE) - An adverse event is any untoward medical experience or change of an existing 
condition that occurs during or after treatment, whether or not it is considered to be related to the 
protocol intervention.  
Reporting Non-serious Adverse Events – Adverse events will be collected after the patient is given the 
study treatment or any study related procedures. Adverse events will be monitored by the PMT. 
Adverse events that do not meet the criteria of serious OR are not unanticipated problems will be 
reported only in the PMT Report. 
Serious Adverse Event (SAE) [Modified from the definition of unexpected adverse drug experience in 21 
CFR 312.32] - defined as any expected or unexpected adverse events that result in any of the following 
outcomes: 
 
Death 
 
Is life-threatening experience (places the subject at immediate risk of death from the event as it 
occurred) 
 
Unplanned hospitalization (equal to or greater than 24 hours) or prolongation of existing 
hospitalization 
 
A persistent or significant disability/incapacity 
 
A congenital anomaly/birth defect 
 
Secondary malignancy 
 
Any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the 
outcomes listed above (examples of such events include allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasias of convulsions that do 
not result in inpatient hospitalization, or the development of drug dependency or drug abuse). 
 
Reporting Serious Adverse Events - begins after study treatment or any study related procedures.  All 
SAEs occurring during this study, whether observed by the physician, nurse, or reported by the patient, 
will be reported according to the approved City of Hope’s Institutional policy [policy effective date:  
05/14/14].  Serious Adverse Events that require expedited reporting will be submitted electronically 
using iRIS. 
7.5 
Adverse Event Name and Severity 
The PI will determine the adverse event name and severity (grade) by using the CTCAE version 4.03.  
Expected Adverse Event - Any event that does not meet the criteria for an unexpected event, OR is an 
expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of 
the research participant experiencing the adverse event. 
Unexpected Adverse Event [21 CFR 312.32 (a)] – An adverse event is unexpected if it is not listed in the 
investigator’s brochure and/or package insert; is not listed at the specificity or severity that has been 


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observed; is not consistent with the risk information described in the protocol and/or consent; is not an 
expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of 
the research participant experiencing the adverse event. 
7.6 
Adverse Event Attribution 
The following definitions will be used to determine the causality (attribution) of the event to the study 
agent or study procedure. 
Definite - The AE is clearly related to the investigational agent or study procedure and unrelated to 
any other cause. 
Probable - The AE is likely related to the investigational agent or study procedure and unlikely 
related to other cause(s). 
Possible -The AE may be related to the investigational agent or study procedure and may be related 
to another cause(s). 
Unlikely -The AE is doubtfully related to the investigational agent or study procedure and likely 
related to another cause(s). 
Unrelated -The AE is clearly not related to the investigational agent or study procedure and is 
attributable to another cause(s). 
7.7 
COH Held IND  
Serious Adverse Events meeting the requirements for expedited reporting to the Food and Drug 
Administration (FDA), as defined in 21 CFR 312.32, will be reported as an IND safety report using the 
MedWatch Form FDA 3500A for Mandatory Reporting. 
The Office of IND Development and Regulatory Affairs (OIDRA) will assist the PI in reporting the event to 
the Food and Drug Administration (FDA). 
The criteria that require reporting using the Medwatch 3500A are: 
 
Any unexpected fatal or life threatening adverse experience associated with use of the drug 
must be reported to the FDA no later than 7 calendar days after initial receipt of the information 
[21 CFR 312.32(c)(2)] 
 
Any adverse experience associated with use of the drug that is both serious and unexpected 
must be submitted no later than 15 calendar days after initial receipt of the information [21 CFR 
312.32(c)(1)] 
 
Any follow-up information to a study report shall be reported as soon as the relevant 
information becomes available. [21 CFR 312.32(d)(3)] 
 
The PI or designee will be responsible for contacting the Office of IND Development and Regulatory 
Affairs (OIDRA) at COH to ensure prompt reporting of safety reports to the FDA.  OIDRA will assist the PI 
with the preparation of the report and submit the report to the FDA in accordance with the approved 
City of Hope’s Institutional policy [policy effective date:  05/14/14]. 
7.8 
Deviations and Unanticipated Problems 
Deviation - A deviation is a divergence from a specific element of a protocol that occurred without prior 
IRB approval.  Investigators may deviate from the protocol to eliminate immediate hazard(s) for the 
protection, safety, and well-being of the study subjects without prior IRB approval.  For any such 
deviation, the PI will notify the COH DSMC and IRB within 5 calendar days of its occurrence via iRIS in 
accordance with the Clinical Research Protocol Deviation policy [policy effective date:  11/07/11].   


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7.9 
Single Subject Exception (SSE) 
An SSE is a planned deviation, meaning that it involves circumstances in which the specific procedures 
called for in a protocol are not in the best interests of a specific patient.  It is a deviation that is 
anticipated and receives prior approval by the PI and the IRB.  The SSE must be submitted as a “Single 
Subject Exception Amendment Request” via iRIS in accordance with IRB guidelines and the Clinical 
Research Protocol Deviation policy [policy effective date:  11/07/11].  An IRB approved SSE does not 
need to be submitted as a deviation to the DSMC. 
 
Unanticipated Problem (UP) – Any incident, experience, or outcome that meets all three of the 
following criteria: 
 
1. Unexpected (in terms of nature, severity, or frequency) given the following: a) the research 
procedures described in the protocol-related documents such as the IRB approved research 
protocol, informed consent document or Investigator Brochure (IB); and b) the characteristics of 
the subject population being studied; AND 
2. Related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcomes may have been caused by the 
drugs, devices or procedures involved in the research); AND 
3. Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm) than previously known or recognized. 
 
Any UP that occurs during study conduct will be reported to the DSMC and IRB in accordance with the 
City of Hope’s Institutional policy [policy effective date:  05/14/14] using iRIS.   
8.0 
AGENT INFORMATION. 
8.1 
Leflunomide 
Leflunomide is an FDA approved agent for the treatment of rheumatoid arthritis. Please refer to the 
Package Insert for additional details not provided in this section. 
8.1.1 
Description and classification 
The chemical name for leflunomide is N-(4’-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide. It 
has a molecular formula C H F N O, a molecular weight of 270.2. Leflunomide is a pyrimidine synthesis 
inhibitor. 
8.1.2 
Mode of action 
Leflunomide is an isoxazole immunomodulatory agent which inhibits dihydroorotate dehydrogenase (an 
enzyme involved in de novo pyrimidine synthesis) and has antiproliferative activity. Several in vivo and 
in vitro experimental models have demonstrated an anti-inflammatory effect. 
8.1.3 
Toxicology 
See Section 6.1.1. 


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8.1.4 
Pharmacology 
Following oral administration, leflunomide is metabolized to an active metabolite teriflunomide (A77 
1726) which is responsible for essentially all of its activity in vivo. Following oral administration, peak 
levels of the active metabolite occurred between 6 to 12 hours after dosing. Due to the very long half-
life of teriflunomide (~2 weeks), a loading dose of 100 mg for 3 days was used in clinical studies to 
facilitate the rapid attainment of steady-state levels of teriflunomide. Without a loading dose, it is 
estimated that attainment of steady-state plasma concentrations would require nearly two months of 
dosing. Biliary recycling is a major contributor to the long elimination half-life of teriflunomide. The 
active metabolite of leflunomide is eliminated slowly from the plasma. Use of a drug elimination 
procedure (cholestyramine or activated charcoal) is used to reduce the drug concentration more rapidly 
after stopping leflunomide therapy. 
8.1.5 
Storage and stability 
Leflunomide tablets should be stored at 25°C (77°F); excursions are permitted to 15–30°C (59–86°F). 
Protect from light. 
8.1.6 
Preparation 
Leflunomide is commercially available for oral administration as tablets containing 10, 20, or 100 mg of 
active drug. 
8.1.7 
Agent administration 
Leflunomide is administered orally. Participants will be given a study calendar to track all doses taken or 
missed. There will be an initial loading dose of 100 mg per day for the first 3 days of doses. Participants 
will then receive leflunomide at the assigned dose level. See Section 5.5.1 for administration details.  
8.1.8 
 Availability and supply 
Tablets in 10 and 20 mg strengths are packaged in bottles of 30 pills per bottle. Leflunomide will be 
provided to patients by the study as an investigational agent. A commercial supply will be purchased and 
distributed as an investigational agent by the Investigational Drug Pharmacy at City of Hope. 
8.2 
Cholestyramine 
Cholestyramine is not the study agent being evaluated for efficacy, but is required for the elimination of 
the study agent leflunomide. Cholestyramine is FDA approved for hypercholesterolemia. 
Cholestyramine use is indicated in the leflunomide package insert to eliminate the leflunomide 
metabolite.  
8.2.1 
Description  
Cholestyramine is a strong anion exchange resin comprised of a quaternary ammonium group attached 
to an inert styrene-divinylbenzene copolymer. Cholestyramine removes bile acids from the body by 
forming insoluble complexes with bile acids in the intestine, which are then excreted in the feces.  
8.2.2 
Other names  
Questran®, Questran Light, Cholybar, Olestyr 
8.2.3 
Mode of action 
Cholestyramine resin adsorbs and combines with the bile acids in the intestine to form an insoluble 
complex which is excreted in the feces. This results in a partial removal of bile acids from the 
enterohepatic circulation by preventing their absorption. 


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8.2.4 
Toxicology 
See Section 6.0. 
8.2.5 
Storage and stability 
Store between 20º-25ºC (68º-77ºF). [See USP Controlled Room Temperature]. Excursions permitted to 
15º-30ºC (59º-86ºF). 
8.2.6 
Agent administration 
Cholestyramine should be administered orally after being prepared per package insert (powder diluted 
in fluid). See Section 5.5 for study administration details.  
8.2.7 
Availability and supply 
Cholestyramine will be provided to participants by the study. A commercial supply will be purchased and 
distributed by the pharmacy at City of Hope. 
9.0 
CORRELATIVE/SPECIAL STUDIES 
9.1 
Pharmacokinetics 
All participants will undergo serial blood sampling to evaluate the steady-state systemic exposure to 
teriflunomide (A77 1726) the active metabolite of leflunomide. Pharmacokinetic sampling will consist of 
trough level monitoring throughout the entire period of active treatment with leflunomide.  
9.1.1 
Specimen Collection 
Prior to initiation of drug administration on day 1 of cycle 1 of treatment and then at follow-up visit 
thereafter (see Table 10 Study Activity Calendar), venous blood will be collected into one 4-mL purple-
top (EDTA) tube. Patients will be instructed not to take their leflunomide dose until after they have had 
their blood drawn on the days of their clinic appointment. Blood samples will be kept on ice until 
prompt delivery (so that samples can be processed within an hour of collection) to the Analytical 
Pharmacology Core Facility (APCF) for processing. Samples are to be taken to Shapiro room 1042. The 
APCF (Leslie Smith-Powell or her representative) will be notified in advance of sampling (ideally at least a 
day in advance) by e-mail to LSmith-Powell@coh.org. Any updates to the location of the laboratory or 
the contact information will be distributed to the study team.  
9.1.2 
Initial specimen processing and storage 
Specimen processing and storage will occur at the APCF plasma is separated from whole blood by 
centrifugation at 1500 x g (within 1 hour). Plasma will then be transferred to appropriately labeled 
polypropylene tubes and stored at < -70C until analysis. Each sample will be labeled with the 
participant’s name, medical record number, date of collection, and actual sample collection time.  
9.1.3 
Analytical Method 
Total and free teriflunomide (A77 1726) plasma concentrations will determined in the Analytical 
Pharmacology Core Facility (APCF) at the City of Hope using a validated LC-MS/MS method. The method 
is based on a previously reported assay that has been validated over a wide dynamic concentration 
range of 10-4000 ng/mL from a starting plasma volume of 200 µl [41].  
9.1.4 
Pharmacokinetic Data Analysis 
Non-compartmental pharmacokinetic analyses of teriflunomide (A77 1726) will be used to determine 
the average steady-state total and free teriflunomide trough concentration (Ctrough). Ctrough results will be 
summarized within and between participants using means and standard deviations. 


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9.2 
Pharmacodynamics 
All participants who undergo serial blood sampling to evaluate pharmacokinetics will also have 
peripheral blood mononuclear cells (PBMC) isolated from their blood samples for exploratory analyses 
of potential pharmacodynamic (PD) markers of the effects of the active metabolite of leflunomide. 
Potential PD assays include the quantitative analysis of intracellular levels of dihydroorotate, orotate, as 
well as nucleotide pools as direct indicators of DHODH inhibition. Furthermore, the expression of 
lymphocyte cell surface activation markers will be assessed as an indirect measure of drug effect. 
Additional PD assays may also be included based on the results of the ex vivo mechanistic studies to be 
performed under 9.4 below. 
9.2.1 
Specimen Collection 
Venous blood will be collected into two 4-mL purple-top (EDTA) tubes. Please refer to Sec 9.1.1 for other 
details of collection. 
9.2.2 
Initial specimen processing and storage 
Any blood remaining in the three 4-mL purple-top tubes used to prepare plasma for PK above will be 
diluted 1:1 with Hank’s Balanced Salt Solution (“HBSS”, Irvine Scientific, Cat. 9228 or equivalent)  and 
combined in a sterile 50 ml conical centrifuge tube. PBMC will then be isolated from the combined 
whole blood sample by Ficoll-gradient separation as described below; 
o Allow Accuspin-Histopaque tubes (“Accuspin”, Sigma Cat. A6929 or A0561, for 12 or 100 tubes, 
respectively) and HBSS to warm to room temperature. Place a Mr. Frosty container in the 
refrigerator and prepare the freezing media by adding 10% DMSO to fetal calf serum and chill at 
4˚C or on ice.  
o Prepare Accuspin tubes by centrifuging at 1000 x g for 1 minute at room temperature (RT) with 
brakes on. Each tube can process up to 20 mLs of whole blood; prepare the appropriate amount of 
tubes necessary. After centrifugation, the Histopaque reagent should be below the barrier of the 
tube. Add 5 mLs of HBSS to the Accuspin tube. Add up to 20 mls of whole blood to each Accuspin 
tube until all the blood has been distributed.  
o Centrifuge the blood sample at 800 x g for 15 minutes at RT with brakes on LOW. After 
centrifugation, three layers should be visible above the barrier of the tube: the plasma layer at the 
top, a cloudy layer in the middle where the PBMC are, and a clear Histopaque reagent layer right 
below. Using a pipette, remove the upper plasma layer to within 2 cm of the cloudy interphase. 
Carefully pipette the cloudy PBMC interphase and transfer to a sterile 50 mL centrifuge tube.  
o Add HBSS up to the 45 mL mark in the centrifuge tube with the PBMC and spin at 400 x g for 10 
minutes at RT with brakes on. Decant the supernatant and loosen the cell pellet before adding 
HBSS to the 45-mL mark again for a second wash. Centrifuge at 300 x g for 10 minutes at RT with 
brakes on. Decant the supernatant, loosen the cell pellet and then add a known volume of HBSS to 
resuspend the cells for counting. Mix the cell suspension up and down with a pipette several times 
before removing a small aliquot for cell count. 
o Centrifuge the cell suspension one final time at 300 x g for 10 minutes at RT with brakes on. PBMC 
should be frozen down at 0.5 – 1 x 107 cells/vial. Determine the volume of freezing media (fetal 
calf serum with 10% DMSO) needed to give a 1 x 107 cell/mL suspension. After the last 
centrifugation is complete, discard supernatant and loosen the cell pellet before adding freezing 
media slowly, a small volume at a time with mixing in between (vortex at low speed). Aliquot 0.5 - 
1 mL of the final cell suspension into individually labeled cryovials. Transfer the cryovials into Mr. 
Frosty and store at -80˚C. Twenty four hours later, cryovials will be transferred to liquid nitrogen 
tanks for long-term storage.  


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9.2.3 
Analytical Method 
The intracellular levels of dihydroorotate, orotate, and nucleotide pools will be performed using liquid 
chromatography tandem mass spectrometry as previously described[42, 43]. Expression of cell surface 
markers of lymphocyte activation (CD25 and CD134) will be measured by flow cytometryas previously 
described[44]. 
9.3 
Pharmacogenomics 
All participants will undergo genetic studies to evaluate germline genetic polymorphisms in the coding 
exome. The potential association between germline polymorphisms in CYP1A2, CYP2C19, and DHODH 
and either toxicity or response will be analyzed. CYP1A2 and CYP2C19 are major leflunomide metabolic 
enzymes and DHODH is the main molecular target of leflunomide 
9.3.1 
Specimen Collection and Processing and Analysis 
Cells will be obtained from the PK processing procedure (Section 9.1.2) for DNA extraction by the APCF 
(one sample per participant). The extracted DNA material will then be sent to the Integrative Genomics 
Core (ICG) for sequencing. 
9.3.2 
Gene Sequencing Data Analysis 
Gene sequences provided by the IGC will be assessed for gene polymorphisms identified in the literature 
[20, 21] associated with leflunomide response in RA.  
9.4 
Ex vivo and Molecular Studies 
9.4.1 
Sample Collection, Transfer, and Initial Processing 
Please see the Appendix for detailed procedures. Any updates to the laboratory locations or contact 
information will be distributed to the study team. Bone marrow specimens will be collected prior to 
drug administration, on day 1 of cycle 2 (all phase II patients and phase I patients enrolled at the 
anticipated RP2D), at progression (where applicable), and at confirmation of CR/sCR (where applicable). 
See Table 10, Study Activity Calendar. Bone marrow aspirate (10 ml) will be collected into a sodium 
heparin (green-top) tube. Samples will be gently inverted 8 times to mix the sample and then promptly 
taken at room temperature to Tinisha McDonald (Kaplan CRB, RM 3016) in Dr. Guido Marcucci’s 
laboratory (or her representative) for processing (so that samples can be processed within an hour of 
collection). Tinisha McDonald (tmcdonald@coh.org) as well as the supervisors Allen Lin (alin@coh.org) 
and Dr. Guido Marcucci (gmarcucci@coh.org) will be notified in advance of sampling (at least 1 day in 
advance) by email. Each sample will be labeled with the participant’s name, medical record number, 
date of collection, and actual sample collection time.  
The Marcucci laboratory will conduct Ficoll-Paque density centrifugation and preparation of 
mononuclear cells (MNCs) and then transfer the sample on ice to Dr. Ralf Büttner at the Rosen 
laboratory (Kaplan CRB, room 1026) for enrichment CD138-positive (CD138+) plasma cells by magnetic 
cell sorting, according to the manufacturer’s instructions. The CD138+ MNCs will be re-suspended in 
complete RPMI medium, counted and stored on ice for further processing.  
9.4.2 
Sample Storage and Distribution for Further Analysis 
At least 5 million (≥5%) CD138-positive MNCs can be expected from each BM aspiration after 
immunomagnetic enrichment. For RNA and DNA analysis without further ex vivo treatment, 500,000 
cells (RNA) and 1,000,000 cells (DNA) will be pelleted and frozen at -80 C. The remaining CD138-
enriched cells will be stored in the vapor phase of a liquid nitrogen tank in freezing medium if the cells 
are not processed immediately. A small aliquot of the cell population (~50,000 cells) will also be used to 
confirm that the enriched cell population is >95% pure. Up to three million CD138-enriched cells will be 


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used for ex vivo cell culture studies either immediately after enrichment or after thawing of the frozen 
cells. See Table 9.3.2 for allocation of samples to downstream assays. If insufficient cell numbers are 
collected for the proposed ex vivo and molecular studies, we will prioritize as follows: 
mRNAseq/miRNAseq > ex vivo cytotoxicity studies > DNA methylation sequencing studies > ex vivo 
synergy studies. 
 
 


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Table 9.3.2 Distribution of CD138-enriched bone marrow aspirate cells for research 
 
Ex vivo teriflunomide or control treatment 
 
No ex vivo treatment 
 
Cytotoxicity 
Synergy 
analysis 
mRNA/miRNA 
profiling 
 
mRNA/miRNA/DNA 
methylation profiling 
Baseline/screening 
X** 
X** 
X*** 
 
X*** 
1 mo. of leflunomide 
 
 
 
 
X*** 
Progression* 
 
 
 
 
X*** 
Complete response* 
 
 
 
 
X*** 
*Where applicable, ** both the phase I and phase II components, *** all participants treated at the 
R2PD (phase II and phase I/II participants)  
 
9.4.3 
Ex vivo Cytotoxicity and Synergy Analysis 
All cells will be grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1x 
antibiotics/antimycotics. Primary pre-treatment MM patient-derived CD138+ cells will be co-cultured 
with increasing concentrations of teriflunomide. Teriflunomide will be used because conversion of 
leflunomide to the active metabolite teriflunomide happens primarily in the intestine. Therefore, we 
consider it unlikely that conversion will occur ex vivo/in vitro, and that use of teriflunomide is the better 
option. Proliferation and cytotoxicity of primary MM cells will be measured based on quantitation of the 
ATP present in cells, MTS assays, and Annexin V staining; IC50 values will be calculated. The cells will also 
be cultured with increasing amounts of teriflunomide and selected MM drugs. Examples from each class 
include the HDAC inhibitor panobinostat, the IMiDs lenalidomide and pomalidomide, the proteasome 
inhibitors ixazomib and bortezomib, the steroid hormone dexamethasone and the CD38 antibody 
daratumumab. To evaluate synergy, we will generate combination index (CI) values by combining 
teriflunomide and the FDA-approved agents at constant ratios based on the IC50s, as described 
previously by Chou TC [45], and generate isolobograms to identify the ratios producing maximal synergy.  
9.4.4 
High Throughput Genomics Analysis 
We will evaluate MM cells treated with teriflunomide ex vivo (as described in Section 9.3.3) and 
leflunomide in vivo (comparison of MM samples from baseline, after one month of treatment and at PD 
or CR, where applicable). DNA (from 1 million cells, in vivo treatment only) and RNA (from 100,000 cells, 
ex vivo and in vivo treatment) will be extracted according to the manufacturer’s instructions for RNA 
isolation (Qiazol, Qiagen) and DNA isolation (DNAzol, Thermofisher) and submitted to the COH 
Integrative Genomics Core for analysis. Sequencing will be performed on an Illumina Hiseq 2500. Image 
and data processing will be conducted using Illumina's pipeline, as well as alignment tools such as 
TopHat (mRNA), Novoalign (miRNA), and Bismark (DNA methylation). Custom R scripts will be used for 
further processing, pathway analysis, and inter-group comparisons. Genetic loci/transcripts identified by 
bisulfite sequencing (DNA-methylation) and RNAseq (mRNA/miRNA sequencing) will be further 
validated using real time RT-PCR assays.  
 
 


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10.0 STUDY CALENDAR 
Table 10 describes required procedures. All procedures may increase in frequency if clinically indicated 
or oriented following toxicity. Adjustments to the treatment cycle due to a hold in study agent are 
detailed in Section 5.2. 
Table 10.0 Study Activity Calendar 
 
 
Scree
n 
-ing a 
Cycle 1, 2, 3 
Cycle 
4+ 
End of Treatment e 
Active 
Follow-
Up j 
Long 
Term 
Follow-
Up k 
Day  
1b 
Day 
14c 
Day 
1d 
Day 
0 f 
Days 
1-11g 
Day 
14h 
Day 
30 i 
Informed Consent l 
X u 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Criteria m 
X 
 
 
 
 
 
 
 
 
 
Registration n 
X n 
 
 
 
 
 
 
 
 
 
Medical history o 
X 
 
 
 
 
 
 
 
 
 
Physical exam -. 
X 
X 
 
X 
 
 
 
X 
 
 
Vital signs p 
X 
X 
 
X 
 
 
 
X 
 
 
Adverse events assessment q 
 
X 
X 
X 
X 
 
 
X 
 
 
Concomitant meds review-. 
X 
X 
 
X 
X 
 
 
X 
 
 
ECOG status r 
X 
X 
 
X 
 
 
 
X 
 
 
Pregnancy test s 
X 
 
 
 
 
 
 
 
 
 
TB antigen test t 
X 
 
 
 
 
 
 
 
 
 
Hepatitis A, B, C testing-. 
X u 
 
 
 
 
 
 
 
 
 
Pulmonary function test -. 
X u 
 
 
 
 
 
 
 
 
 
Extramedullary disease survey v 
X u 
 
 
X w 
X w 
 
 
X w 
X w 
 
Skeletal survey x 
X u 
 
 
 
 
 
 
 
 
 
Clinical bone marrow 
aspiration/biopsy with research 
use of leftover samples 
X u,y 
X y,z, aa 
 
X y,z 
X y,z 
 
 
X y,z 
X y,z 
 
Research bone marrow 
aspiration 
 
X bb 
 
 
 
 
 
 
 
 
CBC with differential cc 
X 
X 
X 
X 
 
 
 
X 
X 
 
Chemistry panel dd 
X 
X 
X 
X 
 
 
 
X 
X 
 
Myeloma labs ee 
X 
X kk 
 
X 
X 
 
 
X 
X 
 
Response assessment ff 
 
X aa 
 
X 
X 
 
 
X 
X 
 
Research blood samplesgg 
 
X 
X 
X 
X 
 
 
 
 
 
Teriflunomide clinical testing hh 
 
 
 
 
X 
 
X ii 
X ii 
 
 
Leflunomide administration jj 
 
Daily 
 
 
 
 
 
 
Cholestyramine g 
 
 
 
 
 
X g 
 
 
 
 
Survival 
 
 
 
 
 
 
 
 
 
X k 
 
a. All screening procedures to be performed within 21 days of start of study agent except informed consent, 
acute hepatitis panel, skeletal survey, bone marrow biopsy, and pulmonary lung function test which may 
occur within 30 days of start of treatment (footnote u) and except for registration which must occur within 7 
days from start of treatment (footnote n).  


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b. Day 1 assessments to be performed within 5 days (120 hours) prior to Day 1 drug (except footnote u for C1 
only). Day 1 safety assessments must be resulted and reviewed prior to administration of agent for the cycle 
that is initiating. Screening assessments, if performed within this aforementioned time frame, may serve as 
C1D1 assessments. Note: items with footnote “aa” are not performed for C1D1; the windows for screening 
assessments still apply. 
c. 
Day 14 evaluations have +/- 3 day window. 
d. Day 1 assessments to be performed within 5 days (120 hours) prior to Day 1 drug. Day 1 safety assessments 
must be resulted and reviewed prior to administration of agent for the cycle that is initiating. 
e. Reasons to end study treatment include disease progression and unacceptable toxicity. See Section 5.8 for 
all criteria.  
f. 
End of Treatment (EOT) Day 0 is defined as the day the determination to end treatment is made. EOT Day 0 
assessments to be performed as soon as feasible, especially in the event of off treatment due to 
unacceptable toxicity and no later than 7 days after decision to end treatment; assessments performed after 
last dose of study agent(s) and within 7 days of the decision to end treatment may serve as EOT Day 0 
assessments.  
g. 
EOT Day 1 is defined as the day cholestyramine administration begins; it will usually follow immediately 
after EOT Day 0, although it will not be deviation if this does not occur. Cholestyramine (8 grams three times 
daily for 11 days) will be administered according to section 5.7.  
h. EOT Day 14 assessments must occur after completion of cholestyramine, and may occur on Day 14 +/- 3 day 
window. If the cholestyramine administration period is extended beyond 11 consecutive days, the EOT Day 
14 assessments may occur outside of this defined window. 
i. 
EOT Day 30 visit has a +/- 2 day window, however this window may be adjusted without deviation to ensure 
that it corresponds to the final teriflunomide clinical test (see footnote ee). All participants will be followed 
until resolution or stabilization of any serious adverse events occurring during treatment or starting within 
30 days of last study drug administration or within the time to complete the EOT Day 30 assessments, which 
every occurs later.  
j. 
Active follow-up will occur for participants who have completed EOT procedures and yet to demonstrate 
disease progression. The active follow-up visit will take place every 28 days (+/- 5 days) from the day of the 
EOT Day 30 visit. Active follow-up will continue until an alternative myeloma therapy has commenced or 
until disease progression.  
k. 
Long term follow-up will occur approximately every 3 months (+/- 14 days) from the EOT Day 30 visit date 
for survival status. Information will be obtained by reviewing the City of Hope medical record, contacting 
the participant and/or a review of outside medical records. Note: this is not intended to be an all-inclusive 
list.  
l. 
Informed consent process to be fully documented: e.g. prospective participant had sufficient time for 
deliberation, all questions were answered, treatment options provided by MD, full study reviewed including 
risks, and a copy of signed consent given to participant.  
m. Inclusion/exclusion criteria are detailed in Section 3.  
n. See Section 4.3 for slot reservation and registration process. Treatment must begin within 7 days of 
registration. Documentation providing Investigator’s confirmation that all eligibility criteria are met must be 
available prior to registration. 
o. Medical history to include review treatment history for myeloma, medical history pertaining to eligibility, 
and demographic information. 
p. Vital signs: Weight, heart rate, blood pressure, respiration rate, temp. Height required only at baseline. 
q. Adverse event (AE) reporting begins for events that occur after start of study agent. AE recording and 
reporting will continue until the completion of the EOT Day 30 visit or until resolution or stabilization of any 
serious AE occurring before the completion the EOT Day 30 assessments, which every occurs later.  
r. 
See Appendix A for ECOG performance status criteria. 
s. 
Serum or urine pregnancy test for women of child bearing potential only. 


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t. 
T-Spot or other TB antigen test. 
u. Consent, acute hepatitis panel, skeletal survey, bone marrow biopsy, bone marrow aspiration, and 
pulmonary lung function test to be completed within 30 days prior to registration.  
v. 
Extramedullary disease survey required at screening for all participants with known or suspected extra-
medullary disease. May include CT scan of the abdomen/pelvis, CT or x-ray of the chest, ultrasound of the 
liver/spleen or abdomen. Technique used at screening/baseline (if applicable) should be used throughout 
the study and include tumor measurements. 
w. Extramedullary disease survey required after screening/baseline only in patients with extramedullary 
disease present at screening/baseline. To occur every 12 weeks or upon clinical suspicion of progressive 
disease.  
x. 
Skeletal survey (including skull, all long bones, pelvis and chest) with tumor measurements (if 
plasmacytomas are present). To be performed at baseline/screening and when clinically indicated. 
y. 
 Leftover bone marrow aspirate from screening/baseline, confirmation of complete response (CR/sCR, when 
applicable) and confirmation of progressive disease (PD, when applicable) (Section 9.4). Bone marrow 
aspirate will be collected into a 10mL or equivalent sodium heparin (green top) tube. Samples will be gently 
inverted approximately 8 times to mix the sample and then promptly taken at room temperature to Tinisha 
McDonald (Kaplan CRB, RM 3016) in Dr. Guido Marcucci’s laboratory for processing (so that samples can be 
processed within an hour of collection). Tinisha McDonald (tmcdonald@coh.org) as well as the supervisors 
Allen Lin (alin@coh.org) and Dr. Guido Marcucci (gmarcucci@coh.org) should be notified in advance of 
sampling (at least 1 day in advance) by email. Changes in laboratory contact information will not be 
considered protocol deviations.  
z. 
Perform bone marrow biopsy and/or aspirate once to confirm suspected CR, sCR, or PD. 
aa. Bone marrow and response assessments are not to be taken on Cycle 1 Day 1; screening labs will be used as 
baseline values. 
bb. Bone marrow aspiration for research on day 1 of cycle 2 (Section 9.4). Note: In the event that the research 
prep for the baseline bone marrow aspirate (footnote y) yields no useable product, this procedure will not 
be performed. In the event that this planned sample collection coincides with confirmation of suspected CR, 
sCR, or PD, then the clinical leftover samples (see footnotes y and z) will be used instead of a research-
specific collection. See contact information for research sample transfer in footnote y. 
cc. CBC with differential: erythrocytes (RBC), hemoglobin, hematocrit, platelets, total WBC plus absolute 
differential counts (neutrophils, lymphocytes, monocytes, eosinophils, basophils).  
dd. Serum chemistry panel: Comprehensive metabolic panel (sodium, potassium, chloride, carbon dioxide, 
creatinine, urea nitrogen, calcium, glucose, albumin, total bilirubin, alkaline phosphatase, total protein, 
ALT/SGPT, AST/SGOT) LDH, magnesium, phosphorus, and uric acid. 
ee. Myeloma labs to include serum protein electrophoresis and immunofixation, quantitative immunoglobulins, 
serum free light chains and 24-hour urine studies (total protein, urine protein electrophoresis and 
immunofixation). Note: 24-hour urine studies to be performed at baseline for all participants, and after 
baseline to confirm complete response or in patients with urine-only measureable disease.  
ff. See Section 11.1 for response assessment criteria. 
gg. Research blood sample collection will occur at the time of blood collection for clinical labs. Venous blood 
will be collected into three 4-mL purple top (EDTA) tubes, gently agitated, placed on ice and brought 
promptly (so that the sample can be processed within the hour) to the APCF in Shapiro room 1042. Notify 
the APCF (Leslie Smith-Powell or her representative) by e-mail to LSmith-Powell@coh.org in advance of 
sampling (ideally at least a day in advance). Discarded cells from one sample will contribute to 
pharmacogenomic studies (Section 9.3.1). Changes in laboratory contact information will not be considered 
protocol deviations.  
hh. Teriflunomide clinical blood assay to be ordered as “leflunomide metabolite” miscellaneous clinical test. 
ii. 
Clinical testing must demonstrate teriflunomide plasma levels less than 0.02 mg/L or 0.02 µg/ml by two 
separate tests at least 14 days apart. If plasma levels are higher than 0.02 mg/L or 0.02 µg/mL, additional 
cholestyramine treatment will be considered.  


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jj. Participants will receive leflunomide orally on a daily basis which will be recorded on the pill diary. The first 
3 loading doses will be given at a dose of 100 mg, after which treatment will continue at a dose of 20 or 10 
mg daily. See administration details in Section 5.5.1. NOTE: On study visit days, participants should refrain 
from taking leflunomide until after blood is collected.  
kk. Myeloma labs may be repeated at C1D1 per investigators discretion and used for disease response 
assessment 
11.0 EVALUATION CRITERIA/MEASUREMENT OF EFFECT 
11.1 Phase I/Primary Endpoint: Toxicity 
The primary endpoint is toxicity. Toxicity will be graded according to the NCI-Common Terminology 
Criteria for Adverse Events version 4.03. Dose limiting toxicity (DLT) is defined in section 13.1 of the 
protocol. The MTD will be based on the assessment of DLT during cycle 1. All patients who are not 
evaluable for dose limiting toxicity will be replaced. 
11.2 Phase II/Primary Endpoint: Response 
The primary endpoint is response. Response will be categorized using the IMWG response criteria for 
multiple myeloma [37, 38]. Table 11.2 summarizes the categories and criteria.  
Table 11.2 Response Criteria 
Response Category 
Response Criteria 
Complete 
response 
(CR)* 
Negative immunofixation of serum & urine, AND 
Disappearance of any soft tissue plasmacytomas, AND 
< 5% plasma cells in bone marrow 
Stringent 
complete 
response (sCR)* 
CR (as defined above) PLUS: 
Normal FLC ratio, AND 
Absence 
of 
clonal 
cells 
in 
bone 
marrow 
by 
immunohistochemistry 
or 
immunofluorescence 
Very 
good 
partial 
response (VGPR)** 
Serum and urine M-component detectable by immunofixation but not on 
electrophoresis, OR 
≥ 90% reduction in serum M-component plus urine M-component < 100mg per 24h 
Partial 
response 
(PR)** 
≥ 50% reduction of serum M protein and reduction in 24h urinary M protein by ≥ 90% 
or to < 200mg per 24h 
If serum and urine M protein are unmeasurable, a ≥ 50% decrease in the difference 
between involved and uninvolved FLC levels is required in place of the M protein 
response criteria 
If serum and urine M protein are unmeasurable, AND serum free light assay is also 
unmeasurable, a ≥ 50% reduction in bone marrow plasma cells is required in place of M 
protein, provided baseline percentage was ≥ 30%. 
In addition to the above criteria, if present at baseline, ≥ 50% reduction in the size of 
soft tissue plasmacytomas is also required 
Minimal 
Response 
 25% but ≤ 49% reduction of serum M protein and reduction in 24-hour urine M-
protein by 50 to 89% which still exceeds 200 mg per 24 hr 


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Response Category 
Response Criteria 
(MR)**, *** 
 In addition to the above criteria, if present at baseline, 25-49% reduction in the size 
of soft tissue plasmacytomas is also required 
 No increase in size or number of lytic bone lesions (development of compression 
fracture does not exclude response) 
Stable disease (SD)** 
Not meeting criteria for CR, VGPR, PR, or PD 
Progressive 
disease 
(PD)** 
Increase of 25% from lowest response value in any one or more of the following: 
 Serum M-component (absolute increase must be ≥ 0.5 g/100ml), and/or 
 Urine M-component (absolute increase must be ≥ 200mg per 24h), and/or 
 Only in patients without measurable serum and urine M-protein levels:  
 The difference between involved and uninvolved FLC levels (absolute 
increase must be > 10mg/dl) 
 Bone marrow plasma cell percentage (absolute % must be ≥ 10%) 
 Definite development of new bone lesions or soft tissue plasmacytomas OR definite 
increase in the size of existing bone lesions or soft tissue plasmacytomas 
 Development of hypercalcemia (corrected serum calcium > 11.5mg/100ml) that can 
be attributed solely to the plasma cell proliferative disorder 
* Confirmation with bone marrow assessment is required for CR and sCR. Only a single bone marrow 
assessment is needed. CR and sCR require no known evidence of progressive or new bone lesions if 
radiographic studies are performed. 
** The following response categories: VGPR, PR, MR, SD, and PD require 2 consecutive assessments 
made at any time before the institution of any new therapy. VGPR, PR, MR, and SD require no known 
evidence of progressive or new bone lesions if radiographic studies are performed. The second 
assessment might be at the time of a planned assessment or earlier, based on the judgement of the 
treating physician.  
*** Based on the recommendations in Rajkumar et al. [38], MR will be reported separately and will be 
distinguished from PR or better. 
11.3 Phase II/Secondary Endpoints: 
Best Overall Response: 
Based on the International Myeloma Working Group (IMWG) criteria, best overall response is the best 
response (sCR/CR/VGPR or PR) recorded from the start of treatment until disease progression. 
Confirmation of sCR/CR/VGPR or PR assessed by IMWG criteria. 
Response Duration: 
Defined as the time interval from the date of first documented response (sCR/CR/VGPR or PR) to 
documented disease progression or death whichever occurs first. 
Clinical Benefit Response: 
Based on the International Myeloma Working Group (IMWG) criteria, clinical benefit response is the 
best response (sCR/CR/VGPR/PR/MR or SD) recorded from start of treatment until disease progression. 
Confirmation of sCR/CR/VGPR/PR/MR or SD assessed by IMWG criteria. 
Overall Survival: 
Defined as the time interval from date of first dose of study drug to date of death from any cause. 
Progression-Free Survival: 


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Defined as the time interval from date of first dose of study drug to first documented disease 
progression or death from any cause, whichever occurs first. 
12.0 DATA REPORTING/PROTOCOL DEVIATIONS 
12.1 Data Reporting 
12.1.1 Confidentiality and Storage of Records 
Electronic Data Collection will be used for this protocol. The data will be stored in encrypted, password 
protected, secure computers that meet all HIPAA requirements. When results of this study are reported 
in medical journals or at meetings, identification of those taking part will not be disclosed. Medical 
records of subjects will be securely maintained in the strictest confidence, according to current legal 
requirements. They will be made available for review, as required by the FDA, HHS, or other authorized 
users such as the NCI, under the guidelines established by the Federal Privacy Act and rules for the 
protection of human subjects. 
12.1.2 Subject Consent Form 
At the time of registration, the original signed and dated Informed Consent form, HIPAA research 
authorization form, and the California Experimental Subject’s Bill of Rights (for the medical record) and 
three copies (for the subject, the research record, and the Coordinating Center) must be available. All 
Institutional, NCI, Federal, and State of California requirements will be fulfilled. 
12.1.3 Data Collection Forms and Submission Schedule  
All data will be collected using electronic data collection, stored as indicated in Section 12.1.1, and will 
be submitted according to the timelines indicated in Table 12.1.3. 
Table 12.1.3 Data Submission Schedule 
Form 
Submission Timeline 
Eligibility Checklist 
Complete prior to registration  
On Study Forms 
Within 14 calendar days of registration 
Baseline Assessment Forms 
Within 14 calendar days of registration 
Treatment Forms 
Within 10 calendar days of treatment administration 
Adverse Event Report Forms 
For cycle 1 only, within 7 calendar days of AE 
assessment/notification; for all other cycles, within 10 calendar 
days of AE assessment/notification  
Response Assessment Forms 
Within 10 calendar days of the response assessment 
Other Assessment Forms (concomitant 
medications) 
Within 10 calendar days of the assessment 
Off Treatment/Off Study Forms 
Within 10 calendar days of end of treatment/study 
Follow up/Survival Forms 
Within 14 calendar days of the protocol defined follow up visit 
date or call 
Eligibility Checklist 
The Eligibility Checklist must be completed by a protocol nurse or clinical research coordinator and 
signed by an authorized investigator prior to registering the subject. See Section 4.3 for the registration 
procedure. 


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12.2 Protocol Deviations 
12.2.1 Deviation Policy 
This protocol will be conducted in accordance with COH’s “Clinical Research Protocol Deviation policy”… 
Deviations from the written protocol that could increase patient risk or alter protocol integrity require 
prior IRB approval of a single subject exception (SSE) request. IRB pre-approved SSE protocol 
modifications are considered an amendment to the protocol and not a deviation. The submission of a 
deviation report is not required. 
Brief interruptions and delays may occasionally be required due to travel delays, airport closure, 
inclement weather, family responsibilities, security alerts, government holidays, etc. This can also 
extend to complications of disease or unrelated medical illnesses not related to disease progression. The 
PI has the discretion to deviate from the protocol when necessary so long as such deviation does not 
threaten patient safety or protocol scientific integrity. Examples include, but are not limited to: a) dose 
adjustments based on excessive patient weight; b) alteration in treatment schedule due to non-
availability of the research participant for treatment; c) laboratory test results which are slightly outside 
the protocol requirements but at levels that do not affect participant safety. These instances are 
considered to be deviations from the protocol. A deviation report will be submitted to the DSMC/IRB 
within five days. 
12.2.2 Reporting of Deviations 
All deviations will be reported to the COH DSMC within five days. The DSMC will forward to report to the 
IRB following review. 
12.2.3 Resolving Disputes 
The COH Investigational Drug Service (IDS) cannot release a research agent that would cause a protocol 
deviation without approval by the PI. Whenever the protocol is ambiguous on a key point, the IDS 
should rely on the PI to clarify the issue. 
In situations where there is misperception or dispute regarding a protocol deviation among the persons 
involved in implementing the protocol, it is the responsibility of the PI to resolve the dispute and the PI 
may consult with the DSMC chair (or designee) to arrive at resolution. 


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13.0 STATISTICAL CONSIDERATIONS 
13.1 Definition of Dose-Limiting Toxicity (DLT) 
A dose limiting toxicity (DLT) will be defined as any of the following toxicities that are at least possibly 
related to leflunomide and occur during cycle 1:  
Any toxicity or series of toxicities resulting in: 
 
Fewer than 21 doses during cycle 1 
 
Extension of Cycle 1 > 35 days 
Hematologic: 
 
Grade 4 neutropenia (ANC <500/mm3)  
 
Grade 3 or 4 febrile neutropenia 
 
Grade 4 thrombocytopenia (<25,000/mm3)  
 
Grade 3 thrombocytopenia (<50,000/mm3) with bleeding  
Non- Hematologic: 
 
Grade 4 non-hematologic toxicity, with the following exceptions:  
o Diarrhea that resolves to G3 within 48 hours  
o Vomiting that resolves to G3 within 48 hours 
o Allergic reaction/hypersensitivity, or  
o Electrolyte/metabolic toxicity corrected to <Grade 1 or baseline within 48 
hours 
 
Grade 3 toxicity despite maximal medical therapy lasting > 7 days 
 
Grade 2 or higher ALT (SGPT)/AST (SGOT)/Total Bilirubin elevation lasting > 14 days.  
 
Grade 3 or higher elevation of ALT (SGPT)/AST (SGOT)/Total Bilirubin of any duration.  
 
13.2 Evaluable Participants and Participant Replacement 
All participants will be evaluable for toxicity from the time of their first treatment with leflunomide. 
Evaluable for Dose-Escalation/De-Escalation Criteria: Participants will be considered evaluable for 
dose-escalation criteria if they receive at least 75% of leflunomide and are followed during cycle 1, or 
experience a DLT. All participants who are not evaluable for dose-escalation criteria will be replaced. 
Note: While patients who receive <75% of the intended dose of leflunomide will be considered 
unevaluable for determination of MTD, if a patient discontinues leflunomide due to toxicities prior to 
receiving 75% of the dose, the patient will be considered evaluable and will not be replaced. 
Evaluable for Response: Participants will have their response classified according to the IMWG response 
criteria (See Section 11). All patients included in the study will be assessed for response to treatment, 
even if there are protocol treatment deviations or if the patient is later determined to be ineligible. Each 
patient will be assigned one of the following categories: 1) complete response (CR), 2) stringent 
complete response (sCR), 3) very good partial response (VGPR), 4) partial response (PR), 5) minimal 
response (MR), 6) stable disease (SD), 7) progressive disease (PD), 8) early death from malignant disease, 
9) early death from toxicity, 10) early death due to other cause, or 11) unknown (not assessable, 
insufficient data).  
All of the patients who met the eligibility criteria (with the possible of exception of those who received 
no study medication) should be included in initial analysis of response rate; overall response 
(sCR/CR/VGPR or PR) and clinical benefit response (sCR/CR/VGPR/PR/MR or SD). Patients in response 


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categories 7-11 should be considered to have a treatment failure. Thus, an incorrect treatment schedule 
or incorrect drug administration does not result in the exclusion from the analysis of the response rate. 
In addition, as part of the phase II anti-myeloma activity assessment, a sub-analysis will be performed 
where participants will be considered evaluable for response if they are confirmed eligible, receive at 
least 75% of leflunomide during the first cycle of therapy and have had their disease re-evaluated (phase 
II primary endpoint).  
13.3 Study Design 
13.3.1 Phase I Design 
This single agent phase I dose-escalation trial is designed to determine the MTD and RP2D. The RP2D of 
leflunomide will generally be the MTD, but it may be less than the MTD based on a review of available 
data/cumulative toxicities. Extensive pharmacokinetic and pharmacodynamic studies are also 
incorporated into this protocol and may be helpful in determining the RP2D. 
While the standard dose of leflunomide, 20mg daily, is FDA approved for continuous treatment of 
rheumatoid arthritis, leflunomide has been administered without an increase in the type, frequency or 
severity of adverse events at doses up to 40 mg/day in patients with Wegner’s granulomatosis, and up 
to 60mg/day in a subset of patients with polyoma BK neuropathy to achieve targeted therapeutic levels. 
We will therefore attempt to escalate the dose of leflunomide to three times that approved for RA using 
a modified rolling six dose-escalation/ de-escalation/expansion design, a more conservative version of 
the rolling six design of Skolnik, et al.[1]. The starting dose of leflunomide is 20mg daily, with the 
intention to test up to 60mg daily. Escalation will proceed in increments of 20mg/day (odd numbered 
dose levels); de-escalation will proceed in decrements of 10mg/day. 
Table 13.3.1 Dose Levels  
Dose Level 
Leflunomide Dose* 
-1 
10 mg PO daily 
1 
20 mg PO daily 
2 
30 mg PO daily 
3 
40 mg PO daily 
4 
50 mg PO daily 
5 
60 mg PO daily 
*NOTE: Leflunomide is administered with a loading dose of 100 mg for the 1st three doses. 
 
13.3.2 Phase I Dose Escalation 
This phase I trial will employ a modified rolling six design, a more conservative version of the rolling 6 
design of Skolnik, et al [1]. In this design, at most 3 patients will be under observation for DLT (in cycle 1) 
on the current test dose level at any time. Patients who are not evaluable for dose escalation will be 
replaced. Once each patient completes cycle 1 and passes without a DLT, an additional patient may be 
accrued on that dose level for up to 6 patients. Once 3 patients have completed cycle 1 with no patient 
at that dose level experiencing a DLT, the dose can be escalated. Up to 3 additional patients may be 
treated at the current dose level. Although this design does not require that 6 patients be treated, no 
more than 6 evaluable patients will be accrued to any dose level during the dose finding portion of this 
study. If at any time, the dose level has 1 documented DLT with fewer than 6 evaluable patients, accrual 
will continue until 6 patients are evaluable. Escalation will terminate as soon as two or more patients 


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experience a DLT attributable to the study treatment. MTD will be the highest dose in which ≤ 1/6 
patients experience a DLT. There will be no dose escalation within a patient. These rules are outlined in 
Table 13.3.2. 
Table 13.3.2: Dose Escalation Rules 
Phase II Design 
Following completion of the phase I portion of the study and identification of the RP2D, the phase II 
portion of the study will be initiated. Recent studies evaluating daratumumab, a CD38 monoclonal 
antibody, as monotherapy for relapsed/refractory multiple myeloma were recently published [39, 40] 
and have led to FDA approval of this agent. One study included patients with relapsed multiple myeloma 
treated with at least three lines of therapy while the other included patients treated with at least two 
prior lines of treatment. Treatment with daratumumab as a single agent lead to an overall response rate 
of 29.2% and 36% respectively in each of these studies. In both studies, the agent investigated was well 
tolerated but required IV dosing once weekly for 8 doses, followed by twice-monthly doses for 8 doses 
and then monthly thereafter. We suspect leflunomide to be similarly well tolerated but with a more 
convenient dosing schedule. Because we are including patients who have received at least three prior 
lines of treatment, we will target an overall response rate of 30% for the phase II portion of the study. 
# Patients on Current Level 
Action 
With DLT^ 
Evaluable 
Evaluable + At Risk^ 
0 
0 
1-2 
Accrue next patient at this level* 
0 
0 
3 
Hold accrual 
0 
1 
1-3 
Accrue next patient at this level 
0 
1 
4 
Hold accrual 
0 
2 
2-4 
Accrue next patient at this level 
0 
2 
5 
Hold accrual 
0 
3-6 
3-6 
Accrue next patient at the next higher level*,+ 
1 
1 
1-2 
Accrue next patient at this level 
1 
1 
3 
Hold Accrual 
1 
2 
2 
Accrue next patient at this level 
1 
2 
3-4 
Hold accrual 
1 
3-5 
3-5 
Accrue next patient at this level 
1 
3-5 
6 
Hold accrual 
1 
6 
6 
Accrue next patient at the next higher level* 
2** 
any 
any 
Accrue next patient at next lower level (max 6) 
^: 
DLT: a patient with a documented DLT 
 
Evaluable: a patient who is either fully evaluable for toxicity for the purpose of dose escalations or has a DLT 
 
At Risk: a patient who is on treatment and has not yet passed the evaluation period nor had a DLT 
*:  During the dose-escalation portion, if higher dose level is already closed, the next lower dose will accrue to a 
total of 6 patients, with 2 or higher DLTs requiring further dose de-escalation. 
+: 
Although under this scenario escalating to the next higher dose level is suggested, additional patients can be 
accrued to the current level -up to n=6 patients. 
**:  Patients treated on a higher dose will have their treatment modified to the dose below the dose level with 2 
DLTs, if pending patients have DLT. 


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This single-center, single arm phase II trial will implement a Simon Two-Stage Optimal Design [2, 3].  to 
evaluate the anti-myeloma activity of leflunomide, when given as a single agent, as assessed by overall 
response rate (ORR), in patients with relapsed or relapsed/refractory MM. The phase II portion of the 
study is expected to enroll a minimum of 19 and a maximum of 39 patients. The sample size is based on 
the desire to discriminate a promising ORR of 30% from a disappointing response rate of 15% using a 
type I error rate of 0.10 and power of 80%.  
At stage 1, 19 patients will be entered on the study. If ≤ 3 responses are seen, the study will be 
terminated. If at least 4 patients achieve response, the trial will continue to the second stage. The 6 
patients treated at the RP2D in the phase I portion will be evaluated as part of this phase II group. The 
probability of early stopping after the first stage is 0.68. 
At stage 2, 20 additional patients will be entered. At the end of stage 2, if 9 or more patients respond, 
leflunomide -as single agent therapy, will be considered worthy of further study. If ≤ 8 patients respond 
further investigation is not warranted. 
13.4 Sample Size and Accrual Rate 
Phase I: In general, the total sample size will depend on the number of dose levels evaluated to 
determine the RP2D. Assuming the highest dose tested is well tolerated (3 patients treated on dose 
levels 1 (20mg) and 3 (40mg); 6 patients treated on assumed MTD -dose level 5 (60mg), the phase I 
portion of the study would enroll and treat 12 patients. The expected sample size is 12-18 patients, 
allowing for 6 patients to replace inevaluable/ineligible patients. A maximum of 36 participants could be 
treated if 6 participants are treated at each dose level. A minimum of 6 patients could be treated if 
leflunomide is not well tolerated.  
Assuming one patient is enrolled each month, accrual is expected to be completed in 12-18 months for 
the phase I portion. When the phase II portion begins, it is anticipated that the accrual rate will increase. 
This is a priority trial and the initiation of the phase II portion is likely to coincide with the end of other 
trials, increasing the pool of potential candidates. Therefore, we estimate a rate of 2 patients per month 
for the phase II portion, for an accrual time of 17 months. The expected follow-up time is 24 months, for 
an estimated maximum total study duration of (18+17+24) 59 months, or roughly five years. In the 
event that participants are receiving treatment at the end of the study, additional options for 
continuation, such as compassionate use, will be considered.  With ~150 relapsed or relapsed/refractory 
MM patients treated at COH per year, we do not expect any problems accruing the required patient 
numbers for both phases of the clinical trial within 29-35 months from start of study, and therefore both 
phases of the clinical trial will be conducted solely at COH. 
13.5 Statistical Analysis Plan 
Patient demographic and baseline characteristics, including age, gender, medical history, and prior 
therapy, will be summarized using descriptive statistics. For continuous variables, descriptive statistics 
(number [n], mean, standard deviation, standard error, median (range) will be provided. For categorical 
variables, patient counts and percentages will be provided. 
Analysis: Observed toxicities will be summarized, for all dose levels, in terms of type (organ affected or 
laboratory determination), severity, time of onset, duration, serum concentration of the active 
leflunomide metabolite, probable association with the study treatment and reversibility or outcome.  
The overall response rate and clinical benefit response rate will be calculated, for the initial and sub-
analysis for all dose levels; Clopper Pearson binomial 95% confidence intervals will be calculated for 
these estimates. As part of the initial analysis of response, the overall response rate (patients that have 
confirmed sCR/CR/VGPR or PR) and clinical benefit response rate (patients that have confirmed 
sCR/CR/VGPR/PR/MR or SD) will be calculated among all patients included in the study even if there are 


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protocol treatment deviations or if the patient is later determined to be ineligible. In addition, as part of 
the phase II anti-myeloma activity assessment, sub-analyses will be performed where participants will 
be considered evaluable for response if they are confirmed eligible, receive at least 75% of leflunomide 
during the first cycle of therapy and have their disease re-evaluated. Response rates will also be 
evaluated based on number/type of prior therapy(ies) and serum concentration of the leflunomide 
metabolite (µg/mL). Time to response and survival will be estimated using the product-limit method of 
Kaplan and Meier.  
Pharmacokinetics: Because serum concentration of the metabolite, teriflunomide (A77 1726), can be 
variable, and because some studies have found an association between serum concentration of this 
metabolite and RA response, samples will be collected throughout the study for analysis by the 
Analytical Pharmacology Core Facility (APCF) for an exploratory analysis relating metabolite levels to 
safety and efficacy outcomes. At the conclusion of the study, total and free teriflunomide levels will be 
summarized at 2- and 4-week sampling time points. Descriptive statistics will be used to characterize 
possible inter-patient variability and relationship to dose, toxicity and response for future studies, 
including guidance with respect to RP2D selection. Additional testing and analysis will be done on 
collected samples to assess, in an exploratory manner, the potential association between germline 
polymorphisms in the DHODH gene and either toxicity or response. 
Pharmacogenomics: To evaluate the independent effect of germline polymorphisms, all models will be 
adjusted for risk factors known to be associated with outcomes studied (e.g., age, disease status). 
Multiple comparisons adjustment with false discovery rate (FDR) will be conducted, and variants with 
FDR ≤ 0.05 will be considered significant. We will specifically look at DHODH, CYP1A2 and CYP2C19 gene 
polymorphisms and haplotypes identified in the literature that are associated with leflunomide 
response/toxicity in RA. Functional annotation of the variants will be performed using ANNOVAR. The 
major goal of this analysis is to identify potential SNPs that might affect the responsiveness of 
treatment, other than the miRNA/mRNA/methylation signature. Given the small sample size, we 
consider this analysis as exploratory and may not provide enough power to detect significant 
associations. 
Ex vivo and molecular studies: The IC50 values for teriflunomide in MM cells will be calculated using 
Prism software (GraphPad). These values will be used for correlation analysis to determine the strength 
and linearity of the relationship between the ex vivo response and the response characteristics in vivo. 
The results will also be compared to responses from cell lines.  
For the studies combining teriflunomide with other agents ex vivo, combination Index (CI) values will be 
calculated using the CalcuSyn software program (Biosoft, Ferguson) to determine which of the 
compounds work synergistically, if any. A combination index of < 0.9 indicates synergism, whereas CI = 
0.9 to 1.1 indicates additive effects. CI > 1.1 indicates antagonism. Isobologram diagrams will be 
generated that show the varying combination treatments and the ratio where maximal synergy is 
obtained.  
The miRNA/mRNA expression profile and DNA methylation data will be used to identify the 
miRNA/mRNA and differential methylated regions (DMRs) that show significant differences between 
responders and non-responders after leflunomide treatment. For miRNA/RNA profiling, fold changes >3 
with an FDR <0.05 will be considered significant. Assuming the dispersion of genes across replicates is 
0.2 and minimum average expression is 10 reads per gene, 10 samples in each group (responder vs. non-
responder) should have 89% power to detect a 3-fold difference between groups at an FDR level of 0.05.  
The major goal of RNA-seq/miRNA-seq/RRBS-seq is to predict potential responders based on pre-
treatment genomic profiling. A logistic regression-based algorithm with risk score formula described 
previously [46] will be used to build a classifier for response prediction. Differentially expressed 
miRNA/mRNA and DMRs together with other known clinical risk factors will be included in the 


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multivariable logistic regression model. The performance of the identified classifier will be evaluated 
with leave one out cross validation (LOOCV). Specifically, for each iteration -as part of a random 
selection process, one sample will be left out as the test sample; the remaining samples will be used to 
build a classifier, and the classifier will be used to predict the response status of the test sample. The 
sensitivity and specificity of the classifier will be calculated, as well as receiver operating characteristic 
(ROC) curve and area under the curve (AUC) to evaluate its accuracy.   
Another goal of the genomic profiling is to identify potential new pathways to complement leflunomide 
treatment. We will compare the miRNA/mRNA/methylation profiles between responders and non-
responders. Differentially expressed genes will be used to identify pathways that are activated in non-
responders, which will provide insights into possible complementary therapy. Gene ontology (GO) and 
KEGG pathways will be analyzed using DAVID and GSEA. Pathways and GO that are activated in non-
responders will be sorted by their multiple comparison adjusted p-value. Potential miRNA targets will be 
identified using TargetScan and the enrichment of GO and pathways will be identified as well. The NCI 
connectivity map, an online resource to predict therapeutic efficacy based on pathway based gene 
expression profiles, will also be explored to determine the complimentary therapeutic agents for 
leflunomide. 
14.0 HUMAN SUBJECT ISSUES 
14.1 Institutional Review Board 
In accordance with City of Hope policies, an Institutional Review Board (IRB) that complies with the 
federal regulations at 45 CFR 46 and 21 CFR 50, 56 and State of California Health and Safety code, Title 
17, must review and approve this protocol and the informed consent form prior to initiation of the 
study. All institutional, NCI, Federal, and State of California regulations must be fulfilled. 
14.2 Recruitment of Subjects 
The multiple myeloma subjects will be recruited from patients undergoing treatment for multiple 
myeloma at City of Hope Cancer Center for hematological malignancies. Any recruitment materials will 
be reviewed and approved by the IRB prior to their use to recruit potential study subjects. 
14.3  Study location and Performance Sites 
This study will be performed at COH.  
14.4 Confidentiality  
This research will be conducted in compliance with federal and state of California requirements relating 
to protected health information (PHI). The study will record individual side effects to study treatment, 
and disease status, and these will be linked to the subject’s identity using a coded study number. The 
principal investigator, co-investigators, and laboratory technicians will have access to this information, 
but all information will be treated confidentially. No identifiers will be used in any subsequent 
publication of these results.  
14.5 Financial Obligations and Compensation 
The investigational agent, leflunomide, and cholestyramine will be provided free of charge by City of 
Hope for the duration of the trial.  
The standard of care drug(s) and standard of care procedures provided will be the responsibility of the 
research participant and/or the insurance carrier. The research participant will be responsible for all 


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copayments, deductibles, and other costs of treatment and diagnostic procedures as set forth by the 
insurance carrier. The research participant and/or the insurance carrier will be billed for the costs of 
treatment and diagnostic procedures in the same way as if the research participant were not in a 
research study. However, neither the research participant nor the insurance carrier will be responsible 
for the research procedures related to this study. 
In the event of physical injury to a research participant, resulting from research procedures, appropriate 
medical treatment will be available at the City of Hope to the injured research participant, however, 
financial compensation will not be available. 
The research participant will not be paid for taking part in this study. 
14.6 Informed Consent Processes  
The Principal Investigator or IRB approved named designate will explain the nature, duration, purpose of 
the study, potential risks, alternatives and potential benefits, and all other information contained in the 
informed consent document. In addition, they will review the experimental subject’s bill of rights and 
the HIPAA research authorization form. Research subjects will be informed that they may withdraw 
from the study at any time and for any reason without prejudice, including as applicable, their current or 
future care or employment at City of Hope or any relationship they have with City of Hope. Research 
subjects will be afforded sufficient time to consider whether or not to participate in the research. 
Prospective research subjects who cannot adequately comprehend the fundamental aspects of the 
research study with a reasonable amount of discussion, education and proctoring will be ineligible for 
enrollment. For those subjects who do comprehend the fundamental aspects of the study, consent will 
be obtained and documented, followed by eligibility testing. The research team will review the results of 
eligibility testing and determine if the subject is a candidate for study enrollment.  
 
 


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Shi, J., et al., Population pharmacokinetics of the active 
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Huttemann, M., et al., Total and free plasma concentrations of 
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32. 
Soukup, T., et al., Genetic polymorphisms in metabolic pathways 
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33. 
Hopkins, A.M., et al., Genetic polymorphism of CYP1A2 but not 
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Wiese, M.D., et al., Polymorphisms in cytochrome P450 2C19 enzyme 
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Dimopoulos, K., P. Gimsing, and K. Gronbaek, The role of 
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Sive, J.I., et al., Global hypomethylation in myeloma is 
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Durie, B.G., et al., International uniform response criteria for 
multiple myeloma. Leukemia, 2006. 20(9): p. 1467-73. 
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Rajkumar, S.V., et al., Consensus recommendations for the uniform 
reporting of clinical trials: report of the International Myeloma 
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Lonial, S., et al., Daratumumab monotherapy in patients with 
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Parekh, J.M., et al., Chromatographic separation and sensitive 
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Ruckemann, K., et al., Leflunomide inhibits pyrimidine de novo 
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Huang, M., et al., A77 1726 induces differentiation of human 
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Slauson, S.D., et al., Flow cytometric analysis of the molecular 
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APPENDIX A: ECOG PERFORMANCE STATUS CRITERIA 
ECOG Performance Status Scale  
Grade 
Criteria 
0 
Normal activity. Fully active, able to carry on all pre-disease performance without 
restriction. 
1 
Symptoms, but ambulatory. Restricted in physically strenuous activity, but ambulatory 
and able to carry out work of a light or sedentary nature (e.g., light housework, office 
work). 
2 
In bed <50% of the time. Ambulatory and capable of all self-care, but unable to carry out 
any work activities. Up and about more than 50% of waking hours. 
3 
In bed >50% of the time. Capable of only limited self-care, confined to bed or chair more 
than 50% of waking hours. 
4 
100% bedridden. Completely disabled. Cannot carry on any self-care. Totally confined to 
bed or chair. 
5 
Dead. 
 
 
 


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APPENDIX B. DETAILED METHODS FOR PHARMACOGENOMIC, EX VIVO, AND MOLECULAR STUDIES 
This is a description of the detailed analytical methods at the time of protocol submission. Any changes 
to the procedures will be stored in the laboratory manual of procedures/standard operating procedures 
or in the laboratory research notes. 
Pharmacogenomics 
The extracted DNA material will then be sent to the Integrative Genomics Core (IGC; Director, Xiwei Wu, 
PhD) for sequencing. The DNA samples will be fragmented into 200 bp, and a pre-capture library will be 
generated following Illumina’s recommended protocol. Agilent SureSelect Human all exon V6 probes will 
be used to capture the coding exome. The captured library will be sequenced on an Illumina Hiseq 2500 
for paired-end 100 bp sequencing for a minimum of 100x coverage. The paired-end sequences will be 
aligned to the human genome (hg38) using Novoalign. Only reads aligned to a unique genomic location 
will be kept for further analysis. The aligned reads will be piled up using Samtools. Variants (SNPs and 
short indels) will be identified using VarScan. The quality of variants will be assessed by the concordance 
of variants to the dbSNP database. The prognostic significance of germline polymorphisms on toxicity 
risk/response will be analyzed using univariate and multivariable logistic regression. To evaluate the 
independent effect of germline polymorphisms, all models will be adjusted for risk factors known to be 
associated with outcomes studied (e.g., age, disease status). Multiple comparisons adjustment with 
false discovery rate (FDR) will be conducted, and variants with FDR ≤ 0.05 will be considered significant. 
We will specifically look at DHODH, CYP1A2 and CYP2C19 gene polymorphisms and haplotypes identified 
in the literature  that are associated with leflunomide response/toxicicty in RA. Functional annotation of 
the variants will be performed using ANNOVAR. The major goal of this analysis is to identify potential 
SNPs that might affect the responsiveness of treatment, other than the miRNA/mRNA/methylation 
signature. Given the small sample size, we consider this analysis as exploratory and may not provide 
enough power to detect significant associations.  
Ex Vivo and Molecular StudiesBone marrow aspirates will be promptly taken on ice to Tinisha 
McDonald in Dr. Guido Marcucci’s lab for Ficoll-Paque density centrifugation and preparation of 
mononuclear cells and then forwarded to Dr. Ralf Büttner at the Rosen laboratory for processing. The 
bone marrow aspirate will first be filtered through a 40 µm mesh to avoid coagulated products. The 
filtrate will be carefully layered on top of Histopaque (Sigma cat. # 1077-1) in 50 ml polypropylene 
centrifuge tubes. The sample will be centrifuged (with the brake set to “off”) at 1500 x g for 25 minutes. 
The mononuclear blood cells at the interface between the Histopaque and the top aqueous layer will 
be aspirated into a clean 15 ml polypropylene centrifuge tube using a glass Pasteur pipette.  
The aspirated cells will then be washed twice in ice cold PBS, cells will be counted and placed on ice for 
enrichment of CD138-positive cells. CD138 is a transmembrane protein expressed on the surface of 
plasma cells. CD138-positive cells will be enriched by magnetic cell sorting according to the 
manufacturer’s instructions (CD138 MicroBeads, Miltenyi Biotec Cat# 130-051-301). In brief, 
mononuclear cells obtained from the density centrifugation will be pelleted by centrifugation and 
incubated with CD138 magnetic beads for 15 minutes at 4 C in MACS buffer, washed in MACS buffer 
and subjected to column separation. To increase the purity of the CD138-positive cells, the eluted 
fraction from the first column will be enriched over a second column, washed 1x in PBS, re-suspended in 
complete RPMI medium, counted and stored on ice for further processing. For RNA, DNA, and protein 
analysis of pre-treatment patient samples, 100,000 cells (RNA) and 1,000,000 cells (DNA, protein) will be 
pelleted and frozen at -80 C. The remaining CD138-enriched cells will be stored in the vapor phase of a 
liquid nitrogen tank in freezing medium (10% DMSO, 50% FBS, 40% RPMI-1640) if the cells are not 
processed immediately. A small aliquot of the cell population will also be used to determine purity of 
the enriched cells by flow cytometry using anti-CD138-PE antibody (Miltenyi Biotec, Cat# 130-081-301). 
Purities of >95% will be accepted for further experiments. 


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All cells will be grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1x 
antibiotics/antimycotics. 
1): To determine the ex vivo cytotoxicity of leflunomide toward MM cell lines and primary MM cells. 
Based on our experience, a minimum of one-hundred million mononuclear cells (MNCs) can be expected 
from each bone marrow (BM) aspiration after Ficoll centrifugation. The COH Cytogenetics Core 
Laboratory will obtain a separate BM sample for standard karyotype analysis and to detect genetic 
variations frequently found in MM. At least 5 million (≥5%) CD138-positive MNCs can be expected from 
each BM aspiration after immunomagnetic enrichment. MM cell lines and MM patient derived CD138+ 
cells (20,000 cells per well of a 96-well plate; in triplicate) will be co-cultured in RPMI-1640 medium with 
increasing concentrations of teriflunomide (0 uM, 50 uM, 100 uM, 200 uM, 300 uM, 400 uM) for 24, 48 
and 72h. Proliferation of primary MM cells will be measured based on quantitation of the ATP present in 
cells (CellTiter-Glo Luminescent Cell Viability Assay, Promega). IC50 values will be calculated using 
GraphPad Prism. IC50 values will also be determined by flow cytometry using Annexin V. The results from 
primary MM cells will be compared with the following human MM cell lines RPMI-8226, MM.1S, NCI-
H929 and U266 and the human bone marrow stromal cell line HS-5, which were purchased from the 
American Type Culture Collection  
2) To determine potential additive or synergistic effects when combining leflunomide with other classes 
of FDA-approved drugs. Primary pre-treatment CD138+ MM cells will be cultured in 96-well plates 
(20,000 cells/well) with increasing amounts of teriflunomide and selected multiple myeloma drugs for 
72h in RPMI-1640 medium supplemented with or without conditioned medium derived from HS-5 
stroma cells. Conditioned medium will be used to model the in vivo cytokine stimulation milieu. We will 
first determine the IC50s of each individual agent by adding increasing amounts of each drug to the cell 
culture for 72h, followed by the MTS assay (colorimetric assay, for cell lines) and ATP assay 
(luminescence assay, for primary cells). Once the IC50s are established using GraphPad Prism, we will 
repeat above cell culture experiments by combining teriflunomide and the FDA-approved agents at 
constant ratios based on the IC50s, as described previously by Chou. Combination Index (CI) values will be 
calculated using the CalcuSyn software program (Biosoft, Ferguson) to determine which of the 
compounds work synergistically, if any. A combination index of < 0.9 indicates synergism, whereas CI = 
0.9 to 1.1 indicates additive effects. CI > 1.1 indicates antagonism. Isobologram diagrams will be 
generated that show the varying combination treatments and the ratio where maximal synergy is 
obtained.  
3) To generate a preliminary RNA/miRNA and DNA-methylation signature associated with response of 
MM cells to leflunomide. We will conduct high resolution genomic profiling (RNA-seq and miRNA-seq) to 
evaluate mRNA and miRNA profiles, and we will also conduct Reduced Representation Bisulfite 
Sequencing (RRBS-seq) for evaluation of DNA methylation profiles. We will compare the profiles of 
primary MM cells before and after in vivo leflunomide therapy and before and after ex vivo 
teriflunomide treatment. These results will be compared with in vitro teriflunomide treatment of human 
MM cell lines, in order to generate information about common molecular pathways of drug response. 
RNA and DNA will be extracted from primary patient samples (CD138-enriched) prior to start of 
treatment and 4 weeks into treatment. RNA will also be extracted from MM cell lines and CD138+ 
primary, pre-treatment cells treated with 300 µM teriflunomide (a concentration that can be achieved in 
vivo) or vehicle control for 24h. DNA (from 1 million cells) and RNA (from 100,000 cells) will be extracted 
according to the manufacturer’s instructions for RNA isolation (Qiazol, Qiagen) and DNA isolation 
(DNAzol, Thermofisher) and submitted to the COH Integrative Genomics Core for analysis.  
Illumina Hiseq 2500 will be used to profile total cellular RNA (RNA-seq) and micro-RNA (miRNA-seq). The 
concentration and integrity of RNA samples will be measured using Nanodrop and Bioanalyzer. Poly(A) 
RNA will be enriched with oligo d(T) and reverse transcribed, and cDNA will be used for mRNA library 
preparation following Illumina’s TrueSeq protocol. Briefly, cDNA will be fragmented and ligated to 


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Illumina adapters. The cDNA fragments of 130-280 bp will be isolated from polyacrylamide-urea gels. In 
addition, all small RNAs of 15-52 nucleotides will be selected separately and used to make small RNA 
libraries following the Illumina's protocol. The libraries will be quantified using qPCR and loaded to cBot 
for clustering generation. Sequencing will be performed on a Hiseq 2500, and image processing and 
base calling conducted using Illumina's pipeline. For mRNA-seq, sequence reads will be mapped to the 
human genome (hg19) using TopHat and the frequency of Refseq genes will be counted with customized 
R scripts. For miRNA-seq, sequences will be adapter-trimmed and aligned to hg19 genome using 
Novoalign, and miRNA expression level will be counted as previously described. The raw counts will then 
be normalized and compared using the Bioconductor package “edgeR”. Gene ontology (GO) and 
pathways will be analyzed using DAVID (Database for Annotation, Visualization, and Integrated 
Discovery), an online bioinformatic resource for functional interpretation of large lists of genes. 
Pathways and GO that correlate with response will be sorted by their multiple comparison adjusted p 
value. Potential miRNA targets will be identified using TargetScan and their functional annotation will be 
done using DAVID.  
The DNA Methylation of 5-methylcytosine will be profiled by reduced representation bisulfite 
sequencing (RRBS-seq). Briefly, purified genomic DNA will be digested by the methylation-insensitive 
restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After 
end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 
bp) will be size selected and subjected to bisulfite conversion and PCR amplification. The library will be 
sequenced on an Illumina Hiseq 2500 with paired end 100bp reads. The reads will be aligned to hg19 
genome and methylation levels of each cytosine in the CG context will be summarized using Bismark. 
The methylation levels of each CG will be compared between leflunomide treated and untreated 
samples to identify hyper and hypo-methylated regions (DMRs). The common DMRs across the cell lines 
will be identified and considered as candidate regions. The location of the DMRs relative to coding genes 
will be annotated using R scripts and RefSeq database. The genes with promoter DMRs and also showing 
differential expression by mRNA-seq will be identified and their functions and pathways will be 
identified by DAVID on online annotation tools and KEGG database.