Phase I-II Study of Ruxolitinib (INCB18424) for Patients With Chronic Myeloid Leukemia (CML) With Minimal Residual Disease While on Therapy With Tyrosine Kinase Inhibitors
Phase: PHASE1
Conditions: Chronic Myelogenous Leukemia, BCR-ABL1 Positive, Chronic Myelogenous Leukemia, BCR-ABL1 Positive in Remission, Minimal Residual Disease, Philadelphia Chromosome Positive, BCR-ABL1 Positive Chronic Myelogenous Leukemia
Sponsor: M.D. Anderson Cancer Center

Protocol Page
Phase I-II Study of Ruxolitinib (INCB18424) for Patients with Chronic Myeloid 
Leukemia (CML) with Minimal Residual Disease While on Therapy with Tyrosine 
Kinase Inhibitors
2012-0697
Core Protocol Information
Short Title 
Ruxolitinib for CML with MRD
Study Chair: 
Jorge Cortes
Additional Contact:
Allison Pike
Rachel R. Abramowicz
Leukemia Protocol Review Group
Additional Memo Recipients:
Recipients List
OPR Recipients (for OPR use only)
None
Study Staff Recipients
None
Department:
Leukemia
Phone:
713-794-5783
Unit:
428
Study Manager:
Allison Pike
Full Title: 
Phase I-II Study of Ruxolitinib (INCB18424) for Patients with Chronic 
Myeloid Leukemia (CML) with Minimal Residual Disease While on 
Therapy with Tyrosine Kinase Inhibitors
Public Description:
 
Protocol Type:
Standard Protocol
Protocol Phase:
Phase I/Phase II
Version Status:
Terminated 10/03/2019
Version:
24
Document Status:
Saved as "Final"
Submitted by:
Rachel R. Abramowicz--5/7/2019 9:28:03 AM
OPR Action:
Accepted by:  Amber M. Cumpian -- 5/8/2019 5:48:18 PM
Which Committee will review this protocol?
 The Clinical Research Committee - (CRC)

Protocol Body
 
  

2012-0697 
April 6, 2015 
Page 1 of 41 
Phase I-II Study of Ruxolitinib (INCB18424) for Patients with Chronic Myeloid Leukemia 
(CML) with Minimal Residual Disease While on Therapy with Tyrosine Kinase Inhibitors
Principal Investigator 
Jorge Cortes, MD 
Department of Leukemia 
MD Anderson Cancer Center 
1515 Holcombe Blvd., Unit 428 
Houston, TX 77030 
(713)792-7305
Study Product: 
Ruxolitinib 
Protocol Number: 
2012-0697 
Coordinating Center: 
MD Anderson Cancer Center 
1515 Holcombe Blvd., Unit 428 
Houston, TX 77030 

 
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1. OBJECTIVES 
 
1.1.Primary Objective:  
1.1.1. 
Phase I: To determine the DLT and MTD of the combination of ruxolitinib 
and a tyrosine kinase inhibitor (TKI) in patients with chronic myeloid 
leukemia (CML).  
 
1.1.2. 
Phase II: To determine the clinical activity of the combination of ruxolitinib 
and a TKI in patients with CML in complete cytogenetic remission (CCyR) 
with minimal residual disease (MRD). 
 
1.2. Secondary Objectives:  
1.2.1. 
Phase I 
1.2.1.1. 
To determine the clinical activity of the combination of ruxolitinib 
and a TKI in patients with CML 
1.2.2. 
Phase II 
1.2.2.1. 
To determine the safety of the combination of ruxolitinib and a 
TKI in patients with CML in CCyR with minimal residual disease. 
1.2.3. 
Both phases 
1.2.3.1. 
Determine the overall survival, event-free survival and survival 
free from transformation to accelerated and blast phase.  
 
1.2.3.2. 
Determine the effect of therapy on bone marrow progenitors in 
clonogenic assays 
 
1.2.3.3. 
Investigate the effect of therapy on molecular responses as 
assessed by genomic DNA PCR. 
 
1.2.3.4. 
Determine the effect of therapy on TKI-resistant quiescent 
leukemic Ph+ stem cells (CFSEmax/CD34+) by flow cytometric evaluation of 
activated Crkl and Jak2 
 
1.2.3.5. 
Assess the effect of therapy on self-renewal and/or survival of 
leukemic stem cells by FISH Analysis on colonies 
 
1.2.3.6. 
Assess Ruxolitinib pharmacokinetics (PK) in preselected time 
intervals during co-administration of this agent with TKIs 
 
 
2. BACKGROUND 
 
2.1. Chronic Myeloid Leukemia 
 
Chronic myeloid leukemia (CML) is a pluripotent stem cell disorder characterized by 
the presence of the Philadelphia (Ph) chromosome in the leukemic cells. The Ph 
chromosome results from a (9;22) translocation in which the c-ABL oncogene has 

 
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moved from chromosome 9 into the BCR (breakpoint cluster region) gene on 
chromosome 22, resulting in a chimeric BCR-ABL gene 1-3. This is the causative 
abnormality in CML. The fused gene encodes an 8.5 kb chimeric mRNA 4,5, which is 
translated into a 210-kDa protein 6. This p210 BCR-ABL protein functions as a 
constitutively activated tyrosine kinase and is uniquely present in the leukemic cells of 
CML patients 7. The breakpoint in the BCR gene occurs either between BCR exon b2 
and b3 or between BCR exons b3 and b4. Therefore, in the mature BCR-ABL mRNA, 
either b2 or b3 is spliced to ABL exon a2, which results in two alternative chimeric 
p210 BCR-ABL proteins, with either a B2A2 or B3A2 junction 8.  
 
2.2. Tyrosine Kinase Inhibitor (TKI) Therapy 
 
Imatinib is a low molecular weight phenylaminopyrimidine designed to selectively 
inhibit BCR-ABL tyrosine kinase activity 9, and is now the standard therapy for newly 
diagnosed patients with CML who do not undergo allogeneic stem cell transplant 10. 
Results of the pivotal IRIS trial have shown that over 80% of patients achieve a CCyR, 
with most of these responses being durable. After 7 years of follow-up, the event-free 
survival rate is 81% and the survival free from transformation to accelerated or blast  
phase is 92%.11 For patients who develop resistance or intolerance to imatinib, effective 
therapy with second generation tyrosine kinase inhibitors has been developed. Two of 
these agents (dasatinib and niloitinib) are currently available, and others are under 
development (e.g, bosutinib). Approximately 50% of patients treated with these agents 
after imatinib failure achieve a CCyR, and these responses are also durable in most 
patients. 
 
Several reports have shown that, among patients who achieve a CCyR with imatinib or 
other TKI, those who achieve a MMR (ie, at 18 months from the start of therapy) have 
an improved EFS and survival free from transformation to accelerated or blast phase 
compared to those with CCyR but no MMR (7-year EFS 95% vs 86%, respectively).12 
In this same analysis, none of the patients who had achieved a MMR transformed to 
accelerated or blast phase. Recently, it has been suggested that achieving a complete 
molecular response (CMR) further improves the long-term outcome of patents after 
treatment with imatinib. In one series, patients who had an MMR had a shorter relapse-
free survival (median 44 months) compared to those that achieved CMR (median not 
reached, with a hazard ratio for relapse 11%.13 Similar results were reported from our 
institution, where patients with sustained complete molecular response have an estimate 
EFS at 7-years of 95%, compared to 85% for those with sustained MMR but no CMR, 
and 50% for those with CCyR but no MMR.14 Thus, improving the molecular response 
of patients who have already achieved a CCyR with TKI has become an important goal 
of therapy. It has been suggested that patients who achieve CMR may discontinue 
therapy with imatinib, an important goal for patients. However, discontinuation of 
imatinib among patient who achieve CMR results in relapse in over 50% of patients.15 
Thus additional measures are needed to make treatment discontinuation a safer 
proposition. 
 
2.3. The role of JAK2 in CML 

 
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The JAK family of kinases comprises four proteins (JAK1, JAK2, JAK3, and TYK2) 
that can associate with cytokine receptor subunits, phosphorylate them, and in doing so 
create docking sites on the receptors for binding of SH2-containing proteins. In general, 
JAKs consist of several domains (JH1-JH7), including a tyrosine kinase domain, and 
the functional significance of these domains has been characterized by mutational 
analysis. JAKs are able to associate with the cytokine receptors as well as with each 
other. Dimerization/oligomerization of cytokine receptor subunits as a result of ligand 
binding leads to juxtaposition of JAKs. This results in transphosphorylation and 
activation of their kinase activity and the phosphorylation of downstream signaling 
proteins such as STATs, Src-kinases, and adaptors such as Shc, Grb2, and Cbl.  
 
JAK proteins activate a number of intracellular signaling proteins, among which STATs 
are the best defined. Binding of a cytokine to its receptor rapidly induces tyrosine 
phosphorylation of the cytoplasmic domains of the receptor by activated JAK kinases, 
thus providing a docking site for STAT proteins, which are then phosphorylated. This 
phosphorylation of STATs leads to their homo- or heterodimerization and translocation 
to the nucleus, followed by DNA binding and gene activation. The specificity for STAT 
phosphorylation is determined by the receptor docking sites and not the JAK kinases. 
Also, different STAT proteins have different DNA-binding affinities, resulting in 
activation of specific genes. STATs also interact with other transcription factors such as 
the p300/cyclic AMP-responsive element binding protein family of coactivators to 
activate genes. The transcriptional activity of STATs may also be regulated by the 
phosphorylation of their serine and threonine residues, although the implications of such 
regulation are not known.  
 
STATs mediate diverse and sometimes opposite cellular events affecting growth, 
differentiation, and apoptosis. For example, STATs can mediate both growth arrest and 
cellular proliferation. Specifically, STAT1 mediates the growth-inhibitory effects of 
IFN- , through the induction of the CDKI p21waf1, whereas STAT5 mediates 
proliferative effects of IL-3 and GM-CSF. Similarly, phosphorylation of STAT3 can 
result both in IL-6- and IL-10-induced growth arrest, and in GM-CSF- and IL-3-induced 
proliferation. STATs also modulate cellular differentiation and apoptosis. STAT3 and 
STAT5 mediate the antiapoptotic effects of IL-6 and IL-2, respectively. STAT1 
activates the caspase cascade through up-regulation of Fas and FasL expression in 
response to IFN- . The exact mechanisms underlying these diverse effects are being 
elucidated. 
 
The JAK-STAT signaling pathway is constitutively activated in a spectrum of human 
malignancies including CML, AML and ALL. Jak2 was first reported to be involved in 
Bcr-Abl transformation by Xie et al in 2001.16 The C-terminal domain of Abl within 
Bcr-Abl is involved in complex formation with Jak2 and a kinase-inactive Jak2 mutant 
blocked the colony forming ability of K562 cells.16 In Bcr-Abl positive cells, Jak2 
activation mediates an increase of c-Myc RNA expression and interferes with 
proteasome-dependent degradation of c-Myc protein.17 Importantly, Jak2 inhibition 
induced apoptosis and reduced colony formation in imatinib-sensitive and imatinib-

 
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resistant Bcr-Abl mutant cell lines, and induced apoptosis in CML cells from patients in 
blast phase but not in normal hematopoietic cells.18 Jak2 inhibition is able to overcome 
drug resistance in blast phase.19 Recent studies have suggested that Jak2 activation is 
critical for the survival of the CML leukemic stem cell. Inhibition of Jak2 activity 
significantly reduces LTC-ICs, impairs β-catenin activity, and induces apoptosis of Ph+ 
quiescent hematopoietic stem cells. Ph+ HSC survival/self-renewal requires expression 
but not activity of BCR-ABL1, which is important for PP2A inhibition and Jak2 
recruitment/activation. Other studies using a retroviral mosyse model have not been 
able to confirm this potential synergistic effect.20 However, this concept has not yet 
explored in vivo or in a human setting. 
 
We hypothesize that adding ruxolitinib to a TKI may improve the molecular response 
of patients with CML with CCyR but no MMR. If a significant number of patients 
convert to a CMR, we will explore whether TKI can be safely discontinued with 
minimal risk of relapse. Throughout the study we will measure the effect of therapy 
with ruxolitinib (administered in combination with continuing TKI therapy) on 
numerous correlative pathobiological parameters by performing experimental testing of 
peripheral blood and bone marrow samples (as described in Appendix A of this 
protocol. 
 
2.4. Ruxolitinib 
 
2.4.1. Overview of Ruxolitinib 
Ruxolitinib (also known as INCB018424 phosphate; IND# 109051, NSC# 752295) 
represents a novel, potent, reversible and selective inhibitor of JAK1 and JAK2- STAT 
signaling that is currently under development for treatment of myeloproliferative 
neoplasms (MPNs) and advanced hematologic malignancies.  
 
2.4.1.1.Structure and molecular weight  
 
The chemical name of ruxolitinib is (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-
pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate.  
The chemical structure of ruxolitinib is shown below: 

 
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Molecular formula: C17H18N6●H3PO4 
Molecular weight: 404.36 
2.4.2. Preclinical Studies  
Ruxolitinib inhibited the splenomegaly and morbidity/mortality in mice resulting from 
intravenous inoculation of cells expressing the same mutated JAK2 (V617F) implicated 
in the pathogenesis of the majority of Philadelphia chromosome negative MPNs.  
Ruxolitinib inhibited erythroid colony formation from mononuclear cells derived from 
PV subjects (IC50 of 223 nM compared to 407 nM for normal donors).  Growth factor 
independent colony formation, a unique characteristic of PV and other MPNs, was 
inhibited more potently with an IC50 of 67 nM for ruxolitinib in cells bearing the JAK2 
V617F mutation compared to cells bearing the wild-type JAK2.  
Effects of ruxolitinib noted in 6 month rat and 12 month dog repeat dose toxicology 
studies were primarily those associated with the mechanism of action of ruxolitinib 
(inhibitor of JAK-STAT signaling). Genetic toxicology assessments (evaluations of 
ruxolitinib in the bacterial mutagenicity assay, in vitro chromosome aberration assay, 
and in vivo micronucleus assay) in rats were negative. In safety pharmacology 
evaluations, an adverse decrease in minute volume in a respiratory study in female rats 
only was noted at the highest dose. In a cardiovascular evaluation of ruxolitinib in dogs, 
electrocardiogram (ECG) parameters and ventricular repolarization were unaffected at 
all doses; whereas the compound lowered blood pressure and increased heart rate 
compared to vehicle control at the highest dose evaluated. In embryo-fetal assessments 
in rat and rabbit, maternal toxicity and minimal embryo-fetal toxicity were noted at the 
highest doses evaluated. Ruxolitinib was not teratogenic in either rat or rabbit. No 
effects were noted on reproductive performance or fertility in male or female rats. 
Increases in post-implantation loss were noted at the higher doses.   
More detailed information on pharmacology of ruxolitinib, single and multiple dose 
pharmacokinetic (PK) studies conducted in multiple species and nonclinical safety 
evaluations can be found in the Investigator’s Brochure (IB). 

 
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2.4.3. Clinical Pharmacology of ruxolitinib 
Following oral, single-dose administration of ruxolitinib capsules in the fasted state, 
ruxolitinib was absorbed rapidly, typically attaining peak plasma concentrations within 
1 to 3 hours after administration for all doses.  After attaining Cmax, the ruxolitinib 
plasma concentrations declined with a mean terminal-phase disposition t1/2 of 
approximately 3-5 hours.  The mean ruxolitinib Cmax and AUC increased with 
approximately linear proportionality to dose for the entire dose range evaluated of 5 to 
200 mg. There was no significant food effect on absorption or exposure. A double-
blind, randomized, placebo-controlled, single dose escalation study (INCB 18424-131 
study) has been conducted to investigate the food effect. Tmax, Cmax, AUC in particular 
were determined.  
The main conclusion was that overall magnitude of the food effect on the ruxolitinib 
exposure is not expected to be clinically significant. 
Ruxolitinib is metabolized in the liver by the cytochrome (CYP) P450 metabolizing 
enzyme system, predominantly by the 3A4 isozyme. Systemic exposure of ruxolitinib 
was appreciably increased (AUC 2-fold higher) when given in combination with 
ketoconazole, a potent CYP3A4 inhibitor, with a similar effect observed on the PD 
activity (cytokine-induced STAT3 phosphorylation). CYP3A4 inducers significantly 
decreased the exposure to ruxolitinib, with essentially no difference observed on the PD 
activity (cytokine-induced STAT3 phosphorylation). This suggests that CYP3A4 
induction with rifampin results in metabolism of ruxolitinib to active metabolites which 
also inhibit JAKs.   
Ruxolitinib was given as a single 25 mg dose to subjects with varying degrees of renal 
function (INCB 18424-142 study) and to subjects with varying degrees of hepatic 
dysfunction or with normal hepatic function (INCB 18424-137 study). Mild, moderate 
or severe impairment of renal function had no statistically significant effect on PK or 
PD parameters; subjects requiring dialysis showed decreased ruxolitinib clearance. The 
mean total AUCs of ruxolitinib were 88%, 29% and 66% higher, respectively, in 
subjects with mild, moderate and severe hepatic impairment compared to subjects with 
normal hepatic function. Terminal half-life of ruxolitinib was increased in subjects with 
hepatic impairment by approximately 2-fold compared to healthy controls. The subjects 
with severe hepatic impairment showed modestly protracted PD activity compared to 
the other hepatically impaired subjects who displayed PD activity similar to the healthy 
controls.  
Additional details as to the clinical pharmacology of ruxolitinib may be found in the IB. 
2.4.4. Ruxolitinib Clinical Safety in Healthy Volunteers 
Ruxolitinib has been administered in single or multiple doses to over 200 healthy 
subjects.  In single dose studies, ruxolitinib has been safe and well tolerated with 
adverse events (AEs) generally mild in intensity, reversible and of similar incidence 
following ruxolitinib treatment compared with placebo or with other control treatments.   
In a 10-day multiple dose study, a total of 71 healthy volunteers in 6 cohorts received 
doses of 50 mg qd, 100 mg qd, 15 mg bid, 25 mg bid or 50 mg bid ruxolitinib or 

 
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placebo (Study INCB 18424-132).  Ruxolitinib was well tolerated in the study, with 
most adverse events were reported equally by both ruxolitinib-treated and placebo-
treated subjects.  Neutropenia was noted in 3 subjects receiving the highest dose of 
ruxolitinib, 50 mg bid.  Neutropenia at the Grade 4 level led to study drug 
discontinuation on Day 5 in one subject and was reported as a Serious Adverse Event.  
There was a decline in mean absolute neutrophil count (ANC) and to a lesser extent, 
mean white blood cell count (WBC) values with ruxolitinib doses of 15 mg bid or 
higher.  In general ANC or WBC returned to Baseline levels within 1 to 2 days 
following the last dose of study drug. Doses of 25 mg bid and 100 mg qd were 
determined to be the maximum tolerated doses (MTDs) in this study based on the dose 
limiting toxicity of neutropenia.  
A definitive QT study was carried out in 50 healthy volunteers, evaluating the effects of 
single doses of 25 mg or 200 mg ruxolitinib compared with placebo and 400 mg 
moxifloxacin (positive control).  The overall conclusion is that there appears to be no 
adverse impact on ECG signaling (little change in heart rate, QRS duration, QTcF 
interval, and no clinically significant slight increase in PR interval) with the 
administration of ruxolitinib. 
For additional details related to studies conducted in Healthy volunteers, consult the IB. 
 
2.4.5. Ruxolitinib Clinical Safety and Efficacy in Myelofibrosis 
 
Safety data were extracted from the clinical database (cut-off for COMFORT-II or  
NVSCINC424A2352: 04-Jan- 2011; cut-off for COMFORT-I or INCB 18424-351: 02-
Nov-2010). AEs by preferred term, and AEs leading to discontinuation/dose 
reduction/dose interruption were updated 120 days after the initial cutoff of the clinical 
database. Cumulative suspected unexpected serious adverse reactions (SUSARs) 
reported from any studies, including the Phase III studies were extracted from the 
Novartis safety database (cut-off for SAEs: 02-Aug-2011). 
 
The Phase III population includes MF patients recruited into the two Phase III studies 
(COMFORT-I and –II) presents available safety data collected during the randomized 
treatment phase of those studies. Patients were treated with either ruxolitinib (at starting 
doses of 15 mg or 20 mg b.i.d. and their dose was optimized up or down depending on 
tolerability and efficacy: COMFORT-I; n=155, COMFORT-II; n=146) or placebo 
(COMFORT-I; n=151) or BAT (COMFORT-II; n=73). 
 
All patients treated in these Phase III studies are included in this population, but only 
patients randomized to ruxolitinib are pooled (n=301 patients, ruxolitinib-treated 
population). The extension phases are not included in the Phase III patient population. 
Data obtained for patients randomized to placebo or BAT who crossed over to 
ruxolitinib are not included. 
 
2.4.5.1.Common adverse events 
 

 
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A summary of most frequently (≥5%) reported AEs in the Phase III population 
regardless of study drug relationship by preferred term is presented in the table below. 
 
The comparison of the control groups to the ruxolitinib patients showed that headache 
was more frequent in ruxolitinib-treated patients (13.6% vs. 6.0% on placebo and 5.5% 
on BAT). Most AEs of headache were Grade 1 or 2. Similarly, dizziness (12.0% vs. 
6.6% on placebo and 6.8% on BAT) was more frequent in ruxolitinib-treated patients, 
again mostly Grade 1 or 2. When adjusted for patient-year exposure, the differences are 
still present for headache and dizziness. 
 
Weight increase was also more frequent in ruxolitinib-treated patients than in the 
control groups (9.6% vs. 1.3% on placebo and 1.4% on BAT). Although some of these 
patients had co-reported AEs of edema, many had a past medical history of weight loss 
and the weight gain usually gradually accumulated over the course of one year of 
treatment. The majority of weight gain AEs were Grade 1 and 2. It is worth noting that 
weight gain may be a beneficial effect in patients with MF, given the catabolic nature 
of the disease and the frequency of weight loss reported as a constitutional symptom. 
 
Other preferred terms with increased frequency in the ruxolitinib arms included 
bruising (2.6% vs. 1.3% on placebo in COMFORT-I only), contusion (8.6% vs. 5.3% 
on placebo and 1.4% on BAT), urinary tract infection (7.3% vs. 4.6% on placebo and 
2.7% on BAT), herpes zoster (4.0% vs. 0.7% on placebo and 0% on BAT) and 
flatulence (3.3% vs. 1.3% on placebo and 0% on BAT). 
 
Abdominal pain was more frequent in the control groups than in the ruxolitinib group 
(43% on placebo and 13.7% on BAT vs. 12% on ruxolitinib), as were weight decrease 
(8.6% on placebo and 8.2% on BAT vs. 1% on ruxolitinib), early satiety (8.6% on 
placebo and 0% on BAT vs. 0.3% on ruxolitinib) and splenic infarction (6.0% on 
placebo and 0% on BAT vs. 1.0% on ruxolitinib). 
 
TABLE:  Most frequently reported adverse events regardless of study drug 
relationship by preferred term in Phase III patients (safety set) 

 
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AEs with frequency of ≥5%  
Preferred terms are sorted in descending order of frequency, as reported in the <Total> 
column. 

 
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A patient with multiple occurrences of an AE under one treatment is counted only once in the 
AE category for that treatment. 
A patient with multiple AEs is counted only once in the total row. 
AEs occurring more than 28 days after the discontinuation of study treatment are not 
summarized. 
Includes patients from safety clinical database-1 
In the table above, Study Incyte INCB 18424-351 is also known as COMFORT-I, while  NVS 
study CINC424A2352 is also known as COMFORT-II. 
 
The most frequently occurring Grade 3 and 4 AEs regardless of study drug relationship 
were hematologic including anemia (14%) and thrombocytopenia (8%). Non-
hematologic Grade 3-4 AEs were infrequent and rarely reported more frequently than 
in the control arms. Two patients (0.7%) had febrile neutropenia. In general, the pattern 
of AEs was similar between the two ruxolitinib arms in both studies, although there 
were some differences in frequency for specific AEs. 
 
2.4.5.2.AEs leading to treatment discontinuation, dose interruption or dose 
reduction 
 
In the ruxolitinib-treated Phase III population, the overall frequency of AEs leading to 
study drug discontinuation was 11%. This frequency was similar across both studies. 
None of the AEs leading to discontinuation was reported in more than two patients in 
any group. 
 
In the ruxolitinib-treated Phase III population, the overall frequency of AEs requiring 
dose reduction or interruption was 59.8%. This frequency was higher than in the control 
groups (placebo: 27.2%, BAT: 15.1%). The most frequently reported AEs requiring 
dose reduction or interruption in ruxolitinib-treated patients were thrombocytopenia 
(36.9%), platelet count decreased (7.6%) and anemia (5.6%). The high frequency for 
thrombocytopenia is due to protocol-mandated dose reductions and interruptions. 
Although there were no protocol-specified guidelines for dose reductions secondary to 
anemia, some investigators chose to reduce a patient’s dose in the setting of anemia to 
minimize this particular cytopenia. The frequency of these AEs was higher than in the 
control groups. All other AEs requiring dose reduction or interruption occurred with a 
frequency of 1.3% or less in the ruxolitinib-treated patients. This information is 
summarized in the Tables below: 
 
Summary of adverse events leading to discontinuation, dose interruption or dose 
reduction 
 

 
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In the tables above, Study Incyte INCB 18424-351 is also known as COMFORT-I, while  NVS 
study CINC424A2352 is also known as COMFORT-II. 
 
2.4.5.3.Events suspected to be drug-related (as per investigator’s assessment) 
 
The adverse events reported below were considered at least possibly related by 
individual investigator assessment. In the ruxolitinib-treated patients, the overall 
frequency of AEs (preferred terms, maximum Grade) with suspected relationship to 
study drug was 77.1%. In COMFORT-I, related AEs were higher in the ruxolitinib-
treatment group than in the placebo group (74.2% vs. 55.6%). Similarly in COMFORT-
II, their frequency was higher in the ruxolitinib group than in the BAT group (80.1% 
vs. 19.2%). The majority of related AEs were Grade 1 or 2. 
 
Thrombocytopenia (36.2%) and anemia (27.2%) were the most commonly reported 
related AEs and the frequency was higher in the ruxolitinib arms than in comparator 
arms for both. This information is summarized in the tables below: 
 
Frequent adverse events with suspected study drug relationship by preferred term 
(at least 5% in any group) and maximum Grade in Phase III patients (Safety set) 
 

 
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In the tables above, Study Incyte INCB 18424-351 is also known as COMFORT-I, while  NVS 
study CINC424A2352 is also known as COMFORT-II. 
 
2.4.5.4.Deaths and other serious adverse events 
 
In the Phase III population, there were 34 deaths in total, 27 of which were on-
treatment deaths: 20 deaths in COMFORT-I (9 in the ruxolitinib group, 11 in the 
placebo group) and 7 deaths in COMFORT-II (4 in the ruxolitinib group, 3 in the BAT 
group). The reasons for death (infections, intestinal perforation, disease progression and 
events probably due to disease progression, bleedings events) were similar in the 
ruxolitinib and the placebo groups. 
 
In the ruxolitinib-treated Phase III population, the overall frequency of SAEs was 
28.9%. This frequency was similar across both studies. The most frequently reported 
SAEs in ruxolitinibtreated patients were anemia (4.0%) and pneumonia (3.7%). 
Pneumonia was the only SAE that was reported in more than 5% in any treatment 
group COMFORT-I ruxolitinib group with 6.5% and COMFORT-II BAT group with 
5.5%). When evaluating all lower respiratory tract infection AEs grouped by MedDRA 
higher level group term (MedDRA: Medical Dictionary for Regulatory Activities), 
there was no appreciable difference across the arms of the studies COMFORT-I:  
ruxolitinib 10.3% vs. placebo 7.3%; COMFORT-II: ruxolitinib 13.1% vs. BAT 18%). 
Most other SAEs were reported in three patients or fewer in any group, with the 
following exceptions: in the placebo group, abdominal pain was 

 
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reported as an SAE in six patients (4.0%), and splenic infarction in four patients 
(2.6%); in the ruxolitinib-treated patients, fatigue, gastrointestinal hemorrhage and 
pyrexia were reported in four patients (1.3%) each. 
 
Frequent serious adverse events regardless of study drug relationship by 
preferred term (at least 1% in any group) in Phase III patients (safety set) 
 
 

 
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In the tables above, Study Incyte INCB 18424-351 is also known as COMFORT-I, while  NVS 
study CINC424A2352 is also known as COMFORT-II. 
 
2.4.5.5.Clinical Laboratory Evaluations 
 
In ruxolitinib-treated patients, the most frequent newly occurring or worsening 
hematology abnormality of any Grade was decreased Hgb (81.7%) and decreased 
platelet count (67.4%). The majority of decreased platelet counts were Grade 1 or 2. 
Approximately half of the newly occurring or worsening decreased Hgb was Grade 1 or 
2. These hematology results are consistent with the observed AE profile. The most 
frequently observed Grade 3-4 hematology laboratory abnormalities were anemia 
(40.5%), thrombocytopenia (10.6%) and neutropenia (6.3%). 

 
2012-0697 
 
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A majority of patients entered the pivotal studies with Grade 1 and 2 abnormal Hgb at 
baseline. Those patients receiving ruxolitinib mostly worsened to the grade 
immediately worse than their baseline grade. Few worsened to Grade 4 at any time 
during the study (11% on COMFORT-I and 8.2% on COMFORT-II). 
 
The Kaplan-Meier estimates of time to onset of first episode of Grade 2 or higher Grade 
anemia show that the majority of Grade 2 or higher Grade new onset or worsening 
anemia occurs within the first 3 months of the study with an estimate of 0.66 (0.60, 
0.71) and a median time to onset of 1.45 months (95% CI: 1.41 – 1.87). 
 
The majority of patients entered the pivotal studies with normal or slightly abnormal 
(Grade 1) platelet counts. Those patients receiving ruxolitinib either remained normal 
throughout the study (30.3% and 32.2% of patients in the COMFORT-I and –II studies 
respectively, remained at Grade 0) or had new onset or worsening to Grade 1 or 2; few 
patients had new onset or worsening platelet counts to Grade 3 (9.0%  and 6.2% in the 
COMFORT-I and –II studies, respectively) or Grade 4 (3.9% and 2.1%, respectively). 
 
The majority of Grade 3 or 4 thrombocytopenia occurred within the first 3 months of 
the study. Furthermore, the Kaplan-Meier estimates of time to resolution of first 
episode of Grade 3 or 4 thrombocytopenia show that the median time to resolution was 
2 weeks (95% CI: 1.29– 2.14). 
 
Transfusions of PRBCs are a relatively common occurrence for MF patients, but 
increased rates of anemia resulted in an increase in transfusion requirements for some 
ruxolitinib-treated patients. The COMFORT-I and –II studies are consistent in 
demonstrating an increased frequency of patients requiring transfusion of one or more 
units of PRBCs while on treatment compared with the comparator arm (59.4% vs. 
37.1% for patients randomized to ruxolitinib vs. placebo, respectively, and 51.4% vs. 
38.4% for patients randomized to ruxolitinib vs. BAT, respectively.) However, there is 
a relatively small difference in the mean number of units transfused per month when 
comparing patients randomized to ruxolitinib vs. placebo who received any transfusion 
(0.92 vs. 0.75, respectively) and similarly when comparing patients randomized to 
ruxolitinib vs. BAT who received any transfusion (0.86 vs. 0.91, respectively). Anemia 
led to discontinuation in only one patient (0.3%) across these Phase III studies. 
 
In the ruxolitinib-treated patients, the most frequent newly occurring or worsening 
biochemistry abnormality was increased ALT and gamma Glutamyltransferase (GT). 
No newly occurring or worsening Grade 4 biochemistry abnormality was reported and 
very few Grade 3 abnormalities were observed; in ruxolitinib-treated patients, two 
patients (0.7%) reported Grade 3 increased bilirubin, four patients (1.3%) Grade 3 
increased ALT and two patients (0.7%) Grade 2 increased AST. 
 
Common terminology criteria for adverse events (CTCAE) Grade 1-2  
hypercholesterolemia was also frequent in the ruxolitinib-treated patients (16.6%). No 
CTCAE Grade 3 or 4 hypercholesterolemia were reported. 

 
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There were few abnormal vital signs in the pivotal studies. Vital signs were comparable 
between treatment groups. The only relevant change in any of the parameters includes 
an increase >25% compared with baseline in systolic blood pressure in the ruxolitinib 
arms (15.5% vs. 7.3%, ruxolitinib vs. placebo, respectively in COMFORT-I, and 17.5% 
vs. 10.1%, ruxolitinib vs. BAT, respectively in COMFORT-II). 
 
In the ruxolitinib-treated patients, three patients (1%) presented with a QTcF>500 ms 
compared to one patient (0.7%) on placebo and none on BAT. One ruxolitinib-treated 
patient (0.3%) presented with a QTcF increase from baseline >60 ms compared to six 
patients (4.0%) on placebo and none on BAT. No relevant changes in heart rate were 
observed. No relevant changes in any other parameters were observed except for an 
increase in the PR interval >25% and to a value >200 ms (3.3% for ruxolitinib vs. 0.7% 
for placebo in COMFORT-I and 5.7% for ruxolitinib vs. 1.6% for BAT in COMFORT-
II). The largest increase in QTcF at any timepoint in the combined ruxolitinib group 
was 4.4 ms at Week 24. 
 
2.4.6. Investigational Agent Supply 
 
2.4.6.1 Ruxolitinib will be provided by Incyte as 5 mg, 10 mg, 15 mg or 20 mg 
tablets.  
 
 
2.4.6.2 Unused or expired drug must be returned to MDACC.  Unused or 
expired drug will be disposed per institutional policy. 
 
2.5. Supply of other CML-specific treatments 
Imatinib, dasatinib and nilotinib are commercially available. Product information may be 
found in the product information for each product available through the following links: 
Imatinib: http://www.pharma.us.novartis.com/product/pi/pdf/gleevec_tabs.pdf 
Nilotinib: http://www.pharma.us.novartis.com/product/pi/pdf/tasigna.pdf 
Dasatinib: http://packageinserts.bms.com/pi/pi_sprycel.pdf 
 
 
3. Criteria for Patient Eligibility 
 
3.1. Inclusion Criteria 
 
3.1.1. Patients 18 years or older with Philadelphia chromosome (Ph)-positive or 
BCR/ABL-positive CML (as determined by cytogenetics, FISH, or PCR).   
3.1.2. Patients must be on continuous TKI therapy for management of their 
CML. Any commercially available and FDA- approved TKI can be used, 
i.e., imatinib mesylate (IM), nilotinib (NIL) or dasatinib (DAS). Patients 
may be receiving TKI at entry in the frontline or salvage setting, including 
patients currently on imatinib after -interferon failure or on dasatinib or 

 
2012-0697 
 
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nilotinib after failure to prior therapy including imatinib.  
3.1.3. Patients must have received the current TKI for at least 18 months and not 
have increased their dose in the last 6 months.  
 
3.1.4. For the phase I portion of the study, patients may be included without a 
CCyR provided they remain in chronic or accelerated phase CML and 
have at least a CHR. For the phase II portion of the study patients must be 
in complete cytogenetic remission (CCyR), regardless of the stage of 
disease they had at the time they started therapy with TKI.  
 
 
3.1.5. Patients must have detectable BCR-ABL transcript levels meeting at least 
one of the following criteria: 
 
3.1.5.1. 
Patient has never achieved a major molecular response (MMR, 
as defined by a BCR-ABL/ABL ≤0.1% in the international 
scale (currently equivalent to 0.28 in the MDACC molecular 
diagnostic laboratory), and transcript levels have shown in at 
least two consecutive measures separated by at least 1 month to 
have increased by any value, or 
 
3.1.5.2. 
Achieved a major molecular response which has been lost, with 
an interim increase in transcript levels by at least one-log, 
confirmed in two consecutive analyses separated by at least 1 
month, or 
 
3.1.5.3. 
The patient has received therapy for at least 2 years and lacks a 
sustained major molecular response, or 
 
3.1.5.4. 
The patient has received therapy for at least 5 years and lacks a 
sustained complete molecular response (CMR, defined as 
transcript levels still detectable in the MDACC molecular 
diagnostic laboratory). 
 
 
3.1.6. Patients must not have had a known interruption of TKI therapy of greater 
than 21 consecutive days or for a total of 6 weeks in the 6 months prior to 
enrollment.  
 
3.1.7. Patients must be able to understand and sign an informed consent 
indicating that they are aware of the investigational nature of this study in 
keeping with the institutional policies.  
 
3.1.8. ECOG performance status <2. 
 
3.1.9. Adequate organ function defined as: bilirubin <2x upper limit of normal 

 
2012-0697 
 
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(ULN) (unless associated with Gilbert’s syndrome), and ALT or AST 
<2.5x ULN. 
 
3.1.10. ANC ≥1 x109/L and platelets ≥100 x109/L. 
 
3.1.11. Serum creatinine < 1.5 mg/dL or creatinine clearance greater or equal than 
60 
cc/min 
as 
defined 
by 
the 
Cockcroft-Gault 
Equation: 
Males(mL/min):(140-age)*IBW(kg) / 72*(serum creatinine (mg/dl)); 
Females (mL/min):0.85*(140-age)*IBW(kg) / 72*(serum creatinine 
(mg/dl))  
 
3.1.12. Women of childbearing potential should be advised to avoid becoming 
pregnant while on therapy with Ruxolitinib and for 30 days after the last 
dose and practice effective methods of contraception.  Men should be 
advised not to father a child while receiving treatment with Ruxolitinib 
and for 30 days after the last dose. Effective methods of contraception for 
this study include barrier methods (e.g., condoms, diaphragm); 
spermicidal jelly or foam; oral, depo provera, or injectable hormonal 
contraceptives; intrauterine devices; tubal ligation; and abstinence.  
 
3.2. Exclusion Criteria 
 
3.2.1. For the phase I portion of the study, patients in blast phase. For the phase II 
portion of the study, patients in accelerated or blast phase. 
  
3.2.2. Patients receiving any other investigational agents 
 
3.2.3. Patients who are pregnant or breast-feeding 
 
3.2.4. Patients with clinically significant heart disease (NYHA Class III or IV) 
 
3.2.5. Patients with QTc >480msec. 
 
3.2.6. Patients taking a potent CYP3A4 inhibitor that cannot be changed to an 
alternate drug. 
 
3.2.7. Known or suspected hypersensitivity to ruxolitinib. 
 
3.2.8. Patients with advanced malignant hepatic tumors. 
 
3.2.9. Patients with known active hepatitis B or C, or HIV infection. 
 
3.2.10. Patients with other medical conditions or concomitant medications that in the 
opinion of the principal investigator may interfere with the therapeutic treatment 
 

 
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4. Treatment Plan 
 
4.1. TKI: Patients will continue receiving commercially available TKIs (IM, NIL or DAS) at 
the dose they had been receiving during the last 6 months. 
 
4.1.1. Phase I: For the phase I portion of the trial, only patients receiving 
imatinib will be included first at the dose the patient had been receiving 
for the past 6 months.  
 
4.1.2.  Once the MTD is defined, 6 patients each may be treated with 
dasatinib and nilotinib in combination with the same dose of ruxolitinib 
established as MTD for the combination with imatinib. 
 
4.1.3. The following dose levels will be used for dose adjustments: 
 
Table 1: Dose adjustment levels for TKI 
Dose level 
Imatinib (mg/d) 
Dasatinib (mg/d)  
Nilotinib (mg) 
-5 
N/A 
20 
150 QD 
-4 
200 
40 
200 QD 
-3 
300 
50 
150 BID 
-2 
400 
70 
200 BID 
-1 
600 
80 
300 BID 
0 
800 
100 
400 BID 
 
 
4.2.Ruxolitinib:  
Phase I: patients will be treated in cohorts of 3. The starting dose will be dose level 0 
(5 mg orally, twice daily; Table 1).  
4.2.1. At least 3 patients will be entered at each level. All patients treated at any one 
dose level must have been observed for at least four weeks before escalating to 
the next dose level.  Patients who come off study prior to the first four weeks 
and have not experienced a DLT will be replaced. 
Table 2: Dose adjustment levels for ruxolitinib 
Dose level 
Ruxolitinib (mg, orally, twice daily) 
-1 
5 once daily 
0 
5 
1 
10 
2 
15 
3 (Target dose) 
20 
 
4.2.2. Dose escalation will be done as follows: 
Dose-limiting toxicity in  
Result  
 
 
 
0/3  
 
 
 
Escalate to next level 
 

 
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1/3  
 
 
 
Enter 3 more patients 
 
1/6  
 
 
 
Escalate to next level 
 
2/3 or > 2/6 
MTD exceeded. This dose level will be 
declared the maximally administered dose. 
Expand previous level to include a total of 6 
patients if not already accrued to this 
number.  
 
 
4.2.2.1 
For the phase 1 aspect of the protocol:  Prior to advancing dose 
levels, a cohort summary must be completed and submitted to the 
Clinical Research Monitor in the IND Office. 
 
4.2.3. Definition of Dose-Limiting Toxicity (DLT) 
 
4.2.3.1. 
Dose limiting toxicity will be defined in the first 28 days of therapy. 
 
4.2.3.2. 
The toxicity profile of these agents is well known. Non-hematologic 
DLT will be defined as grade 3 or 4 elevation of ALT or AST 
possibly related to TKI.  
 
 
4.2.3.3. 
Hematologic DLT is defined as grade 4 neutropenia, anemia, and/or 
thrombocytopenia lasting for 4 weeks or more.  
 
4.2.4. If dose-level 0 exceeds MTD, subsequent patients will be treated at dose level -
1. Additional cohorts at lower doses (down to dose level -2) may be included if 
higher doses exceed MTD. If dose level -2 exceeds MTD, the study will be 
terminated.   
 
4.3. Phase II: The target dose for the phase II portion of the study is 20 mg orally twice 
daily, unless MTD is defined at a lower dose level in the phase I portion of the study. 
Other dose levels shown in Table 1 will be used for dose adjustments for toxicity 
during therapy. 
 
4.4. Once MTD is defined for imatinib in the phase I portion of the study the phase 2 portion 
of the study will start with imatinib only. The phase I portion of the study with 
dasatinib and nilotinib will open once MTD is defined for the imatinib-based 
combination.  
 
4.4.1. Opening of phase I for dasatinib and nilotinib may occur 
simultaneously with opening of phase 2 portion of the study for the 
imatinib-based combination.  
 

 
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4.4.2. Once and only when an MTD is defined for dasatinib and nilotinib-
based combinations, patients may be treated with these combinations in 
the phase 2 portion of the study. 
 
4.4.3. When all three combinations are entered into the phase 2 portion of the 
study, patients may be treated with any of these combinations depending 
on the TKI they were receiving prior to entering this study. The 
aggregate response will be calculated for all patients, independent of the 
TKI being received.  
 
4.5. Treatment with ruxolitinib (in both phase I and phase II) will continue uninterrupted 
unless there are indications for dose delays/modifications as shown in Section 4.5. 
 
4.6. Dosing Delays/Dose Modifications 
 
4.6.1. Patients experiencing unacceptable toxicity directly attributable to the study drugs 
should temporarily stop treatment according to the guidelines in the dose 
adjustment schema.  
 
4.6.2. Toxicity grading will be according to the NCI CTCAE, v4. The following 
guidelines for dose adjustment for drug-related toxicities are recommended.  
 
4.6.3. TKI: dose modifications and treatment interruptions will be performed 
according to institutional guidelines and standard clinical practice. 
Briefly, dose adjustments will be as follows: 
 
4.6.3.1. 
Non-Hematologic Toxicity 
4.6.3.1.1. Persistent Grade 2: Patients with persistent grade 2 
toxicity 
that 
is 
considered 
clinically 
significant, 
unresponsive to appropriate therapy, may have treatment 
held until the toxicity has resolved to grade ≤1.  Therapy 
may then be resumed at the same dose the patient was 
receiving at the time treatment was interrupted. If the grade 
2 toxicity recurs, therapy may be held until the toxicity has 
resolved to grade 1. Treatment may then be resumed with a 
one dose level reduction. 
 
4.6.3.1.2. Grade 3-4: If a patient experiences Grade 3-4 toxicity that is 
considered clinically significant and possibly related to TKI, therapy must 
be withheld until the toxicity has resolved to Grade ≤1. Treatment may 
then be resumed with a one dose level reduction. If toxicity recurs, 
additional dose reductions may be implemented following the same 
general guidelines. 
 

 
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4.6.3.2. 
Hematologic Toxicity 
 
4.6.3.2.1. If neutrophils are <0.5 x 109/L and/or platelets are <50 x 
x109/L, hold therapy until granulocytes are above 0.5 
x109/L and platelets are above 50 x109/L, then resume 
therapy at 1 dose level reduction from the dose the patient 
was receiving at the time therapy was interrupted. 
 
4.6.3.2.2. If a similar degree of toxicity returns, a further dose 
reduction by one dose level can be performed, using the 
above procedures.  
 
4.6.3.2.3. There will be no making up for missed doses. 
4.6.4.  
 
4.6.5. Ruxolitinib: dose modifications for toxicity will be done according to the 
following guidelines: 
 
4.6.5.1. 
Non-Hematologic Toxicity 
4.6.5.1.1. Persistent Grade 2: Patients with persistent grade 2 
toxicity 
that 
is 
considered 
clinically 
significant, 
unresponsive to appropriate therapy, may have treatment 
held until the toxicity has resolved to grade ≤1.  Therapy 
may then be resumed at the same dose the patient was 
receiving at the time treatment was interrupted. If the grade 
2 toxicity recurs, therapy may be held until the toxicity has 
resolved to grade 1. Treatment may then be resumed with a 
one dose level reduction. 
 
4.6.5.1.2. Grade 3-4: If a patient experiences Grade 3-4 toxicity that is 
considered possibly related to ruxolitinib, therapy must be withheld until 
the toxicity has resolved to Grade ≤1. Treatment may then be resumed 
with a one dose level reduction. If toxicity recurs, additional dose 
reductions may be implemented following the same general guidelines. 
 
4.6.5.2. 
Hematologic Toxicity 
 
4.6.5.2.1. If neutrophils are <0.5 x 109/L, and/or platelets are <50 x 
x109/L, and/or hemoglobin <8 g/dL, hold therapy until 
granulocytes are ≥0.5 x109/L and platelets are ≥50 x109/L 
and hemoglobin ≥8 g/dL, then resume therapy. 
 
4.6.5.2.2. For neutropenia:  
 

 
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4.6.5.2.2.1.If recovery to 0.5 to less than 1 x 109/L, re-start therapy with two 
dose level reduction for at least 2 weeks; if stable, may escalate the 
dose by one dose level.   
 
If recovery to >1 x109/L, re-start therapy with one dose level 
reduction for at least 2 weeks; if stable, may escalate the dose by 
one dose level.  
4.6.5.2.3. If a similar degree of toxicity returns, a further dose 
reduction by one dose level can be performed, using the 
above procedures.  
 
4.6.5.2.4. There will be no making up for missed doses. 
 
4.6.5.3.For thrombocytopenia:  
 
4.6.5.3.1. If recovery to 50 to <75 x109/L, resume therapy at 5 mg twice daily for 
at least 2 weeks; if stable may increase the dose to 10 mg twice daily. 
 
4.6.5.3.2. If recovery to 75 to 100 x109/L, resume therapy at 10 mg twice daily 
for at least 2 weeks; if stable may increase the dose to 15 mg twice daily. 
 
4.6.5.3.3. If recovery to >100 to 125 x109/L, resume therapy at 15 mg twice 
daily for at least 2 weeks; if stable may increase the dose to 20 mg twice 
daily. 
 
4.6.5.4.For anemia:  
 
4.6.5.4.1.1.If recovery to 8 to less than 10 g/dL, re-start therapy with two dose 
level reduction for at least 2 weeks; if stable, may escalate the dose 
by one dose level.   
 
4.6.5.4.2. If recovery to >10 g/dL, re-start therapy with one dose level reduction 
for at least 2 weeks; if stable, may escalate the dose by one dose level. 
 
4.6.5.5.If more than one cytopenia meets criteria for interruption and dose adjustment, 
use the greatest dose reduction indicated.  
4.6.5.6.Modifications of dose schedules other than the above will be allowed within 
the following guidelines: 
 
4.6.5.6.1. Alternative dose reductions can be made to keep clinically 
significant, ruxolitinib-related toxicity grade ≤2. However, 
the lowest acceptable dose level is -2 and the highest is 
what is established as MTD (and no greater than 20 mg 

 
2012-0697 
 
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twice daily).  
 
4.6.5.6.2. Dose adjustments by more than 1 dose level at a time can 
be considered when judged in the best interest of the patient 
(e.g., neutropenia with sepsis, bleeding requiring platelet 
transfusions) when toxicity has resolved. The reason for 
this reduction will be discussed with the PI and the sponsor 
and documented in the medical record. 
 
4.6.5.6.3. A patient who has had a dose reduction because of any of 
the reasons mentioned above may have their dose re-
escalated provided the patient has remained free of toxicity 
requiring dose adjustments as defined above for at least 1 
month. Escalation will be made by 1 dose-level increments 
only, and not more frequent than every month.  
 
4.6.6. Dose Escalation: Patients that do not achieve an improvement in molecular 
response after 3 months (e.g., from major to complete or from non-major to 
major) and have experienced no grade ≥3 toxicity may have the dose of 
ruxolitinib escalated up to the dose determined to be MTD. 
4.6.6.1.Dose escalation of Ruxolitinib will be done in increments of 5 mg per day at a 
time and no more frequent than once every 4 weeks. Dose escalations will be 
from 5 mg BID, to 5 mg in am and 10 mg in pm (or vice versa), to 10 mg BID, 
to 10 mg in am and 15 mg in pm (or vice versa), to 15 mg BID, to 15 mg in am 
and 20 mg in pm (or vice versa), to 20 mg BID.  
4.6.6.2. Patients who have had a prior dose reduction of ruxolitinib and who have no 
toxicity for at least 1 month of therapy may increase the dose of ruxolitinib 
following the guidelines mentioned above. Dose escalation of the TKI is not 
allowed. 
 
4.6.7. Occasional missed doses of TKI will not be considered a deviation.  Missed dosed 
of ≥ 2 weeks without adequate justification will be considered a protocol 
deviation. 
 
4.6.8. Duration of Therapy: Patients may receive therapy for six months. After 
completion of the 6th month, patients may continue therapy if in the opinion of the 
investigator there is clinical benefit provided there has been no grade 3 or higher 
non-hematologic toxicity attributable to ruxolitinib at the doses being 
administered. In these instances therapy may continue for up to 2 years form the 
start of study therapy.   
 
4.6.8.1. 
Patients who achieve a complete molecular remission sustained for 2 
years with at least 2 PCR assessments per year can hold therapy with 
ruxolitinib and/or TKI and be monitored with PCR. If there is 
reappearance of transcripts detectable by PCR, therapy with one or 
both agents can be resumed at the doses being administered at the time 

 
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treatment was interrupted.  
 
4.6.9. Concomitant Medications 
 
4.6.9.1. 
Patients may not receive any other treatment for CML while on study. 
This includes the use of hydroxyurea and anagrelide. 
 
4.6.9.2. 
The use of other medications for management of comorbidities or 
adverse events is allowed, as clinically indicated.  
 
4.6.9.3. 
Concomitant medications will be captured as part of the medical 
record but will not be entered in the case report form. 
 
4.6.9.4. 
Potent CYP3A4 inhibitors should be avoided whenever possible.  
Concomitant administration of these agents should be discussed with 
the principal investigator. 
 
5. Evaluation During Study 
 
5.1. Pretreatment Evaluation (within 7 days from treatment start, unless otherwise 
specified)   
 
5.1.1. A complete history and physical examination including performance status. 
 
5.1.2. CBC, platelet count and differential (differential not required if WBC <0.5 
x109/L), total bilirubin, SGPT (or SGOT), and creatinine within 1 week. 
 
5.1.3. Bone marrow aspirate for morphology (if not done within 6 months). 
 
5.1.4. Cytogenetic analysis or FISH in bone marrow or peripheral blood (if not done 
within 3 months). 
 
5.1.5. Pregnancy test (blood or urine) for female patients of childbearing potential 
within 7 days before initiation of study drug dosing. 
 
5.1.6. Peripheral blood and/or bone marrow for PCR.  
 
5.1.7. Peripheral blood and/or bone marrow for correlative studies (optional – 
Appendix A, & B).  Not all optional samples may be collected.  
 
5.1.8. EKG 
 
5.2. Evaluation During Study  
 
5.2.1. 
Physical exam and evaluation of toxicity (clinic visit or telephone interview) 
after 1 week (±3 days), 4 weeks (±1 week) from the start of therapy, then every 

 
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3 months (±1 month) for the first 6 months, then every 6 to 12 months. 
 
If the evaluation for toxicity is done by phone, a report of physical exam from 
home physician is acceptable provided there is a physical exam at MDACC at 
least every 6 months (+/- 1 month) during the first year and every 12 months 
(+/- 1 month) thereafter. 
 
5.2.2. 
Through interview, we will review the patient's adherence with therapy (TKI 
and ruxolitinib). 
 
5.2.3. 
CBC, platelet, differential every 1-2 weeks (+/-1 week) for 8 weeks from the 
start of therapy, then every 3 months (±3 weeks). Differential not required if 
WBC ≤0.5 x109/L.  
 
5.2.4. 
Bone marrow aspirate with cytogenetics or FISH every 3-6 months in year 1, 
then as clinically indicated. 
 
5.2.5. 
Total bilirubin, SGPT or SGOT, and creatinine every 1-2 weeks (+/- 1 week) for 
8 weeks from the start of therapy, then every 3 months (±3 weeks). 
 
5.2.6. 
PCR (peripheral blood and/or bone marrow) every 4 weeks (±1 week) for 3 
months, then every 3-6 months until one year, then every 6-12 months. 
 
5.2.6.1.Patients who permanently discontinue therapy with TKI will have PCR every 
4 weeks (±1 week) for the first 6 months after discontinuation of TKI, then 
every 3 months (±3 weeks) for 12 months, then every 6 months (±2 months). 
 
5.2.7. 
Peripheral blood and bone marrow for correlative studies every 3-6 months in 
year 1, then every 6-12 months. Not all optional samples may be collected. 
(Optional – Appendix A & B).   
 
5.2.8. 
Pharmacokinetics (PK) (optional): blood for PK for ruxolitinib at baseline, 0.5, 
2 and 4 h from dosing on days 1, and before your next dose at the 3, 6, 9 and 12 
month (±1 month) visits.   Occasional missed PK samples will not constitute a 
protocol deviation/violation.   
 
5.2.9. 
CBC, blood chemistries, EKG’s may be done at outside facilities.  Under 
exceptional circumstances other tests might be done at outside facilities after 
discussion with PI.  All outside lab results will be faxed or mailed to MDACC 
and reviewed and signed by the investigator. 
 
5.2.10. Patients that come off therapy will continue follow-up for toxicity (clinic visit 
or telephone interview) for 30 days (+/- 5 days) after end of therapy. 
 

 
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* Every effort will be made to collect optional procedures at all time points for all 
patients; however, missing collection in one or more of these time points in 
occasional patients will not be considered a protocol deviation/violation.  
Outside Physician Participation During Treatment 
1. MDACC Physician communication with the outside physician is required prior to the 
patient returning to the local physician. 
*This will be documented in the patient record 
2. A letter to the local physician outlining the patient's participation in a clinical trial 
will request local physician agreement to supervise the patient's care (Appendix EE) 
3. Protocol required evaluations outside MDACC will be documented by telephone, fax 
or e-mail.  Fax and/or e-mail will be dated and signed by the MDACC physician, 
indicating that they have reviewed it. 
4. Changes in drug dose and/or schedule must be discussed with and approved by the 
MDACC physician investigator, or their representative prior to initiation, and will be 
documented in the patient record. 
5. A copy of the informed consent, protocol abstract, treatment schema and evaluation 
during treatment will be provided to the local physician. 
6. Documentation to be provided by the local physician will include drug administration 
records, progress notes, reports of protocol required laboratory and diagnostic studies 
and documentation of any hospitalizations. 
7. The home physician will be requested to report to the MDACC physician investigator 
all life threatening events within 24 hours of documented occurrence. 
8. Patients will return to MDACC every 3 months for 6 months then every 6-12 months 
thereafter for evaluation.  If necessary, patients may have the first 3 month evaluation 
conducted through their local physician if approved by the MDACC physician 
investigator.   
 
6. Criteria for Response 
 
6.1. A favorable response for the primary endpoint is defined as a greater than a one-log 
reduction of BCR-ABL transcript levels from the baseline level or disappearance of 
BCR-ABL transcripts (i.e., includes complete molecular response). 
 
6.2. For the purposes of this study, progression will be defined as a confirmed loss of 
complete cytogenetic response (CCyR) (i.e., >0% Ph+ metaphases among 20 
metaphases counted by karyotype, or >10% positive by FISH) for patients who enter 
the study with this response. “Confirmed” is defined here as assessed in two 
consecutive cytogenetic analyses separated by at least a month.  
 
6.3. Survival endpoints (overall, event-free and free from blastic transformation) will be 
measured from the time of start of ruxolitinib therapy. 
 
7. Criteria for Removal from Study 

 
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7.1. The patient receives CML-directed therapy other than TKI and ruxolitinib. 
 
7.2. The patient develops disease progression defined as confirmed loss of CCyR, confirmed 
loss of CHR or transformation to accelerated or blast phase of CML. 
 
7.3. The dose of TKI is increased beyond the dose being used at the time of study entry or 
new therapies for their leukemia are added.  
 
7.4. The patient has a continuous, non-planned interruption of TKI therapy for greater than 8 
weeks (e.g., for non-compliance). 
 
7.5. The patient withdraws consent. 
 
7.6. The treating physician considers it in the best interest of the patient. 
 
7.7. Pregnancy or suspected pregnancy 
 
8. Investigational Agent (Ruxolitinib) Packaging and Labeling 
 
8.1. Ruxolitinib 5 mg tablets are packaged as 60 count in high-density polyethylene (HDPE) 
bottles. The bottles will include labeling “New Drug - Limited by Federal (USA) Law to 
Investigational Use”.  
8.2. Preparation 
8.2.1. Tablets will be provided to the site and no specific preparation of study 
medication is required prior to administration. 
8.3. Handling and Storage: Investigational product must be dispensed or administered 
according to procedures described herein. Only subjects enrolled in the study may 
receive investigational product, in accordance with all applicable regulatory 
requirements. Only authorized site staff may supply or administer investigational 
product. All investigational products must be stored in a secure area with access limited 
to the Principal Investigator and authorized site staff and under physical conditions that 
are consistent with investigational product-specific requirements. The bottles of tablets 
should be stored at room temperature, 15°C to 30°C (59°F to 86°F). Stability studies will 
be conducted on all clinical batches to support the clinical trial. Any unused or expired 
investigational product will be returned to MDACC to be disposed per institutional 
policy.  
8.4. Importantly, for the purposes of this study, the TKI agents (IM, NIL, DAS) are not 
considered investigational agents and they will be supplied by commercial sources (per 
routine clinical care). 
 
 
9. Adverse Event Reporting 
 
9.1. Adverse events will be documented in the medical record and entered into PDMS/CORe.  
PDMS/CORe will be used as the electronic case report form for this protocol. 

 
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9.2. Adverse event is any untoward medical occurrence that may present during treatment 
with a pharmaceutical product but which does not necessarily have a causal relationship 
with this treatment.  
 
Adverse drug reaction (ADR) is a response to a drug which is noxious and unintended 
and which occurs at doses normally used in man for prophylaxis, diagnosis, or therapy 
of disease or for the modification of physiologic function.  
 
Assessing causal connections between agents and disease is fundamental to the 
understanding of adverse drug reactions. In general, a drug may be considered a 
contributory cause of an adverse event if, had the drug not been administered, 1) the 
event would not have happened at all, 2) the event would have occurred later than it 
actually did, or 3) the event would have been less severe. 
 
The Investigator (or physician designee) is responsible for verifying and providing 
source documentation for all adverse events and assigning the attribution for each event 
for all subjects enrolled on the trial. 
 
9.3. Adverse Events (AEs) will be evaluated according to the latest CTC version and 
documented in medical record.  Only unexpected AEs will be recorded in the Case 
Report Form (CRF).  The Leukemia Specific Adverse Events Recording and Reporting 
Guidelines will be utilized.  Expected events during leukemia therapy are: 
 
9.3.1. 
Myelosuppression-Related Events (due to disease or leukemia therapy) 
 
9.3.1.1. febrile or infection episodes not requiring management in the intensive 
care unit 
 
9.3.1.2. epistaxis or bleeding except for catastrophic CNS or pulmonary 
hemorrhage 
 
9.3.1.3. anemia, neutropenia, lymphopenia, thrombocytopenia, leukopenia 
 
9.3.2. 
Disease-Related Events 
 
9.3.2.1. symptoms associated with anemia 
 
9.3.2.2. fatigue 
 
9.3.2.2.1. weakness 
 
9.3.2.2.2. shortness of breath 
 
9.3.2.3. electrolyte abnormalities (sodium, potassium, bicarbonate, CO2, 
magnesium) 

 
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9.3.2.4. chemistry abnormalities (LDH, phosphorus, calcium, BUN, protein, 
albumin, uric acid, alkaline phosphatase, glucose) 
 
9.3.2.5. coagulation abnormalities 
 
9.3.2.6. disease specific therapy (induction, maintenance, salvage, or stem cell 
therapy) 
 
9.3.2.7. alopecia 
 
9.3.2.8. bone,  joint, or muscle pain 
 
9.3.2.9. liver function test abnormalities associated with infection or disease 
progression 
 
9.3.2.10. disease progression 
 
9.3.2.11. abnormal hematologic values  
 
9.3.3. General Therapy Related Events 
 
9.3.3.1. catheter related events 
 
9.3.3.2. renal failure related to tumor lysis syndrome or antibiotic/ antifungal 
therapy 
 
9.3.3.3. rash related to antibiotic use 
 
9.3.3.4. Hospitalization for the management of any of the above expected 
events 
 
9.4. Abnormal hematologic values will not be recorded on the case report form.  For 
abnormal chemical values, the apogee or nadir (whichever is appropriate) will be 
reported per course on the case report form. 
 
9.5. Serious Adverse Event Reporting (SAE) 
 
Serious Adverse Event Reporting (SAE) for M. D. Anderson-Sponsored IND 
Protocols 
 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of 
either the investigator or the sponsor, it results in any of the following outcomes: 
 Death 

 
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 A life-threatening adverse drug experience – any adverse experience that places 
the patient, in the view of the initial reporter, at immediate risk of death from the 
adverse experience as it occurred. It does not include an adverse experience that, 
had it occurred in a more severe form, might have caused death. 
 Inpatient hospitalization or prolongation of existing hospitalization 
 A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions. 
 A congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or convulsions 
that do not result in inpatient hospitalization, or the development of drug dependency or 
drug abuse (21 CFR 312.32). 
 Important medical events as defined above, may also be considered serious 
adverse events. Any important medical event can and should be reported as an 
SAE if deemed appropriate by the Principal Investigator or the IND Sponsor, 
IND Office. 
 
 All events occurring during the conduct of a protocol and meeting the definition 
of a SAE must be reported to the IRB in accordance with the timeframes and 
procedures outlined in “The University of Texas M. D. Anderson Cancer Center 
Institutional Review Board Policy for Investigators on Reporting Unanticipated 
Adverse Events for Drugs and Devices”. Unless stated otherwise in the protocol, 
all SAEs, expected or unexpected, must be reported to the IND Office, regardless 
of attribution (within 5 working days of knowledge of the event). 
 
 All life-threatening or fatal events, that are unexpected, and related to the study 
drug, must have a written report submitted within 24 hours (next working day) 
of knowledge of the event to the Safety Project Manager in the IND Office.  
 
 Unless otherwise noted, the electronic SAE application (eSAE) will be utilized 
for safety reporting to the IND Office and MDACC IRB.  
 
 Serious adverse events will be captured from the time of the first protocol-specific 
intervention, until 30 days after the last dose of drug, unless the participant 
withdraws consent. Serious adverse events must be followed until clinical 

 
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recovery is complete and laboratory tests have returned to baseline, progression of 
the event has stabilized, or there has been acceptable resolution of the event.   
 
 Additionally, any serious adverse events that occur after the 30 day time period 
that are related to the study treatment must be reported to the IND Office. This 
may include the development of a secondary malignancy. 
 
Reporting to FDA: 
 Serious adverse events will be forwarded to FDA by the IND Sponsor (Safety 
Project Manager IND Office) according to 21 CFR 312.32. 
It is the responsibility of the PI and the research team to ensure serious adverse 
events are reported according to the Code of Federal Regulations, Good Clinical 
Practices, the protocol guidelines, the sponsor’s guidelines, and Institutional Review 
Board policy. 
Investigator Communication with Supporting Companies: 
Serious adverse event will be reported to Incyte via fax to Telerx:  1-866-726-9234. 
 
9.6. Annual Reports 
 
If the FDA has granted an IND number, it is a requirement of 21 CFR 312.33, that an 
annual report is provided to the FDA within 60-days of the IND anniversary date.  21 
CRF 312.33 provides the data elements that are to be submitted in the report.  The 
Annual Report (AR) should be filed in the study's Regulatory Binder, and a copy 
provided to Incyte as a supporter of this study as follows: 
Kathy Lenard Roberts, Executive Director Pharmacovigilance 
Incyte Corporation 
Experimental Station - E400 
RT 141 & Henry Clay Road 
Wilmington, DE 19880 
Bus 302.498.6727 
E-mail:  kroberts@incyte.com 
 
10. Statistical Considerations 
 
10.1. This is a single-arm, open-label, phase I-II trial to evaluate the toxicity and efficacy of 
ruxolitinib in CML patients in complete cytogenetic remission but with minimal 
residual disease while continuing to receive TKI therapy. A total of 48 patients will be 
accrued at a rate of 1 to 2 patients per month.  
 
Patients lost to follow-up or those who stop therapy for personal or financial reasons 

 
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need to be replaced and do not count as failure. Patients who discontinue because of 
unacceptable toxicity are included in the stopping rule for toxicity and will also be 
counted as failures in efficacy calculations. 
 
10.2. Phase I 
 
This is a 3+3 phase I trial to determine the MTD for Ruxolitinib while patients are 
treated with tyrosine kinase inhibitors (TKIs). Below are the 3+3 algorithm rules. 
 
Dose-Escalation Algorithm: 
 Enroll 3 patients at dose level 0. 
 If no DLT is observed in at least 3 evaluable patients during the first cycle, the dose 
will be considered a well-tolerated dose and dose escalation will continue to the 
next dose level. 
 If any DLT is observed in 1 of 3 evaluable patients during the first cycle, additional 
patients will be recruited to expand the cohort up to 6 evaluable patients at the same 
dose level. 
 If any DLT is observed in only 1 of 6 evaluable patients during the first cycle, the 
dose will be considered tolerated and dose escalation will continue to the next dose 
level. 
 If any DLT is observed in ≥ 2 of 3 or 2 of 6 evaluable patients during the first cycle, 
this dose is considered a non-tolerated dose and dose escalation will discontinue 
and the MTD has been exceeded.  
 Once the MTD has been exceeded, treat another 3 patients at the previous dose 
level if there were only 3 patients treated at that dose level. 
 The MTD is the highest dose level at which 6 patients were treated and at most 1 
patient experienced a DLT. 
Since there are only 3 doses to be considered and we will have a maximum of 6 
patients at each dose for each patient group, the maximum number of patients 
enrolled in this phase I trial will be 18 patients. If dose level zero exceeds the MTD 
additional patients may be required for exploration of lower doses. 
 
10.3. 
Phase II 
The part of the study will be a phase II trial with a Bayesian design with early stopping 
rules for toxicity. The phase II portion of the study will use the highest dose level that 
induced DLT in ≤1/6 patients in phase I. A total of 30 patients will be treated in this 
phase II study. The accrual rate is about 2 to 3 patients per month. 
 
The primary endpoint (response) of this trial is to determine if the residual disease as 
measured by PCR can decrease by at least 1 log or become undetectable within 12 
months from the start of study therapy. We will determine the proportion of patients with 
response after 12 months of treatment. In this setting, it would be difficult to do an 
interim monitoring for futility.  Patients who lose CCyR within 12 months will be 
counted as treatment failures. 
 

 
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We plan to recruit 30 CML patients to achieve a 88% power to detect that the difference 
between a proportion of 0.20 response rate is significantly different from 0.05 
(alpha=0.05).  Summary statistics will be used to describe the clinical and demographic 
characteristics of the study population. 
 
10.3.1. Toxicity Monitoring during Phase II  
 
With the concern of treatment related unacceptable toxicity defined as grade 3 or 4 
toxicity that prevents patients from continuing therapy with ruxolitinib despite optimal 
management of adverse events.  The following rules apply for each arm.  Denote the 
probability of toxicity by PE. We assume PE ~ beta (0.2, 1.8).  Our stopping rule is given 
by the following probability statement: Pr(PE > 0.20 | data)  > 0.80 . That is, we will stop 
the trial if, at any time during the study, we determine that there is more than 95% 
chance that the unacceptable toxicity is more than 20%. The study will be stopped early 
if (The number of unacceptable toxicities)/ (The number of patients evaluated) ≥ 4/9, 
5/12, 5/15, 6/18, 7/21, 7/24, 8/27, 9/30. We will apply these stopping boundaries starting 
from the ninth patient and in cohorts of 3. The operating characteristics are summarized 
in table 2. 
Table 2: Operating characteristics for monitoring of toxicities 
 
True Toxicity 
Rate 
Early Stopping 
Probability 
Sample Size 
25th percentile Median 75th percentile 
0.10 
0.020 
30 
30 
30 
0.20 
0.274 
24 
30 
30 
0.30 
0.698 
9 
15 
30 
0.40 
0.940 
9 
9 
15 
0.50 
0.994 
9 
9 
12 
 
References (includes those cited in the Appendix) 
 
1. 
de Klein A, van Kessel AG, Grosveld G, et al. A cellular oncogene is translocated to the 
Philadelphia chromosome in chronic myelocytic leukaemia. Nature. 1982;300:765-767. 
2. 
Heisterkamp N, Stephenson JR, Groffen J, et al. Localization of the c-ab1 oncogene 
adjacent to a translocation break point in chronic myelocytic leukaemia. Nature. 1983;306:239-
242. 
3. 
Rowley JD. Letter: A new consistent chromosomal abnormality in chronic myelogenous 
leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293. 
4. 
Grosveld G, Verwoerd T, van Agthoven T, et al. The chronic myelocytic cell line K562 
contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript. Mol Cell Biol. 
1986;6:607-616. 
5. 
Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr genes in 
chronic myelogenous leukaemia. Nature. 1985;315:550-554. 

 
2012-0697 
 
April 6, 2015 
 
Page 36 of 41 
 
6. 
Ben-Neriah Y, Daley GQ, Mes-Masson AM, Witte ON, Baltimore D. The chronic 
myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene. Science. 
1986;233:212-214. 
7. 
Kloetzer W, Kurzrock R, Smith L, et al. The human cellular abl gene product in the 
chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity. 
Virology. 1985;140:230-238. 
8. 
Shtivelman E, Lifshitz B, Gale RP, Roe BA, Canaani E. Alternative splicing of RNAs 
transcribed from the human abl gene and from the bcr-abl fused gene. Cell. 1986;47:277-284. 
9. 
Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl 
tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566. 
10. 
Peggs K, Mackinnon S. Imatinib mesylate--the new gold standard for treatment of 
chronic myeloid leukemia. N Engl J Med. 2003;348:1048-1050. 
11. 
O'Brien SG, Guilhot F, Goldman J, et al. International Randomized Study of Interferon 
Versus STI571 (IRIS) 7-Year Follow-up: Sustained Survival, Low Rate of Transformation and 
Increased Rate of Major Molecular Response (MMR) in Patients (pts) with Newly Diagnosed 
Chronic Myeloid Leukemia in Chronic Phase (CML-CP) Treated with Imatinib (IM) Blood. 
2008;112:Abst# 186. 
12. 
Hughes T, Hochhaus A, Branford S, et al. Reduction of BCR-ABL Transcript Levels at 
6, 12, and 18 Months (mo) Correlates with Long-Term Outcomes on Imatinib (IM) at 72 Mo: An 
Analysis from the International Randomized Study of Interferon versus STI571 (IRIS) in 
Patients (pts) with Chronic Phase Chronic Myeloid Leukemia (CML-CP). Blood. 
2008;112:Abst# 334. 
13. 
Press RD, Galderisi C, Yang R, et al. A half-log increase in BCR-ABL RNA predicts a 
higher risk of relapse in patients with chronic myeloid leukemia with an imatinib-induced 
complete cytogenetic response. Clin Cancer Res. 2007;13:6136-6143. 
14. 
Verma D, Kantarjian H, Shan J, et al. Sustained Complete Molecular Response to 
Imatinib in Chronic Myeloid Leukemia (CML): a Target Worth Aiming and Achieving? Blood. 
2009;114:208 (Abst# 505). 
15. 
Mahon FX, Huguet F, Guilhot F, et al. Is It Possible to Stop Imatinib in Patients with 
Chronic Myeloid Leukemia? An Update from a French Pilot Study and First Results from the 
Multicentre « Stop Imatinib » (STIM) Study Blood. 2008;112:Abstract# 187. 
16. 
Xie S, Wang Y, Liu J, et al. Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl 
transformation. Oncogene. 2001;20:6188-6195. 
17. 
Xie S, Lin H, Sun T, Arlinghaus RB. Jak2 is involved in c-Myc induction by Bcr-Abl. 
Oncogene. 2002;21:7137-7146. 
18. 
Samanta AK, Chakraborty SN, Wang Y, et al. Jak2 inhibition deactivates Lyn kinase 
through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic 
myelogenous leukemia patients. Oncogene. 2009;28:1669-1681. 
19. 
Samanta AK, Chakraborty SN, Wang Y, Schlette E, Reddy EP, Arlinghaus RB. 
Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes 
Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML). Genes Cancer. 
2010;1:346-359. 
20. 
Traer E, MacKenzie R, Snead J, Druker B, Deininger M. JAK2-Mediated Extrinsic 
Survival of CML Stem Cells: Exploring the Potential Combination of BCR-ABL and JAK2 
Inhibitors In Vivo Blood. 2010:Abstract #199. 

 
2012-0697 
 
April 6, 2015 
 
Page 37 of 41 
 
21. 
Copland M, Hamilton A, Elrick LJ, et al. Dasatinib (BMS-354825) targets an earlier 
progenitor population than imatinib in primary CML but does not eliminate the quiescent 
fraction. Blood. 2006;107:4532-4539. 
22. 
Neviani P, Harb JG, Oaks JJ, et al. BCR-ABL1 Kinase Activity but Not Its Expression Is 
Dispensable for Ph+ Quiescent Stem Cell Survival Which Depends on the PP2A-Controlled Jak2 
Activation and Is Sensitive to FTY720 Treatment Blood. 2010;116(Ash Annual Meeting 
Abstracts) Astract #515. 
23. 
Neviani P, Santhanam R, Ma Y, et al. Activation of PP2A by FTY720 Inhibits Survival 
and Self-Renewal of the Ph(+) Chronic Myelogenous Leukemia (CML) CD34+/CD38– Stem 
Cell through the Simultaneous Suppression of BCR/ABL and BCR/ABL– independent Signals. 
Blood. 2008;112 
189. 
24. 
Graham SM, Jorgensen HG, Allan E, et al. Primitive, quiescent, Philadelphia-positive 
stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood. 
2002;99:319-325. 
25. 
Holyoake T, Jiang X, Eaves C, Eaves A. Isolation of a highly quiescent subpopulation of 
primitive leukemic cells in chronic myeloid leukemia. Blood. 1999;94:2056-2064. 
26. 
Holyoake TL, Jiang X, Jorgensen HG, et al. Primitive quiescent leukemic cells from 
patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro 
in association with up-regulation of expression of interleukin-3. Blood. 2001;97:720-728. 
27. 
Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-macrophage progenitors as 
candidate leukemic stem cells in blast-crisis CML. N Engl J Med. 2004;351:657-667. 
28.  
Ruxolitinib (INCB018424) Investigators' Brochure. Incyte Corporation, Wilmington, DE, 
USA, 2012. 
29.  
Gleevec USPI, Novartis AG, Basel, Switzerland, 2012. 
30.  
Tasigna USPI, Novartis AG, Basel, Switzerland, 2012. 
31.  
Sprycel USPI, Bristol-Myers Squibb, Princeton, NJ, USA, 2012. 
 
 
 
 

 
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Appendix A. Correlative studies (including assessment of stem cell renewal factors) 
 
CORRELATIVE/SPECIAL STUDIES: 
1. Investigate the effect of therapy on molecular responses as assessed by genomic DNA 
PCR. 
 
2. Determine the effect of therapy on TKI-resistant quiescent leukemic Ph+ stem cells 
(CFSEmax/CD34+) by flow cytometric evaluation of activated Crkl and Jak2 
 
3. Assess effect of therapy on self-renewal and/or survival of leukemic stem cells by FISH 
Analysis on colonies  
 
Rationale: 
Given that 1) quiescent Ph+ stem cell (HSC) show innate resistant to TKIs (IM, NIL, DAS) 
monotherapy and, TKIs force proliferating primitive progenitors into quiescence21; and 2) 
we have evidence that BCR-ABL1 expression is essential for recruiting and activating 
JAK2 which, in turn, controls survival and self-renewal of Ph+ CFSEmax/CD34+ 
HSCs22,23, it is plausible that treating TKI-treated CML patients in CMR with a JAK2 
inhibitor (such as ruxolitinib) may result in eradication of CML through induction of 
apoptosis of the quiescent leukemic HSC. 
 
Specifically we will: 
1) Assess changes in the number of leukemic stem cells by BCR-ABL1 genomic PCR in 
BM CD34+ cells before and after ruxolitinib treatment. We do expect a marked decrease 
in the levels of genomic PCR BCR-ABL1 positivity if JAK2 activity controls survival of 
leukemic HSCs. 
2) Determine whether   the ruxolitinib + TKI combination exerts its anti-leukemic activity 
towards Ph+ HSCs by direct suppression of JAK2 activity in the quiescent TKI-resistant 
CML HSCs by CFSE-labelling of CD34+ BM cells before and after treatment and 
monitoring of JAK2 activity by intracellular flow cytometry on the gated CFSEmax cells. 
As controls, pCRKL levels will be also monitored on the same cell fractions. It is 
expected a decreased number of quiescent HSCs expressing both high levels of pJAK2 
and pCrkl (as read-out of BCR-ABL1 activity). 
3)  To formally determine whether suppression of JAK2 by ruxolitinib inhibits self-renewal 
and survival of the primitive leukemic HSC we will assess a) the serial replating colony 
forming efficiency (CFC/replating assay) of CD34+/CD38- cell from untreated and 
Ruxolitinib-treated patients, and b) the frequency of long term culture-initiating cells 
(LTC-IC assays), respectively. D-FISH and/or RT-PCR for BCR-ABL1 will be 
performed on plucked colonies to specifically determine the percentage of Ph+ colonies. 
If JAK2 plays a pivotal role in the regulation of self-renewal and survival of leukemic 
HSC we do expect the emerging colonies to be Ph-negative or to have a marked decrease 
of the Ph-positive colonies. 
 
Methods: 
Tracking of cell division using CFSE staining 

 
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CD34+ cells from CML primary samples will be stained with 0.25µM of carboxyfluorescein 
diacetate succimidyl diester (CFSE, CellTrace CFSE proliferation Kit, Invitrogen). 
CFSE-stained cells were then FACS-sorted to isolate a narrow peak of CFSE 
fluorescence, in order to be able to resolve discreet populations corresponding to cell 
divisions. Non-dividing cells, whose cell cycle is arrested by the addition of Colcemid 
(100ng/ml, Sigma) in the medium, were used to determine the CFSEmax population after 
3 to 6 days in culture. The cells were harvested after 6 days in culture, stained with anti-
CD34 PE (Beckman Coulter), the viability stain 7-AAD (BD Bioscience) to determine 
the number of viable quiescent cells (CFSEmax/CD34+/7-AAD- cells). The calculation 
of the recovery of quiescent cells, expressed as a percentage of the absolute number of 
cells used to establish the culture at time zero, will performed as described in 24.  
 
Intracellular flow cytometry. 
The CFSE+/CD34+ fraction from 3-6 day cultured CFSE-stained CML samples and the will be 
stained with anti-CD34 PE (BD), anti-CD38 PEcy7 (eBioscience) then fixed and 
permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD 
Biosciences). Cells will then stained with either a specific primary antibody or an isotype 
matched control and a secondary goat F(ab’) anti-rabbit conjugated to Alexa Fluor 647 
(Invitrogen). Stained samples will be analyzed on a LSRII flow cytometer (BD). The 
primary antibodies used will be: rabbit anti-pCRKL (pY207) and rabbit anti-phospho-
JAK2 (pY1007/1008) (Cell Signaling Technology). 
 
Long-term culture-initiating assays (LTC-IC assay) 
2x106 bone marrow cells from CML patients will be cultured on 35mm dishes with a 1:1 mix of 
irradiated (80Gy) M2-10B4 (producing IL-3 and G-CSF) and SI/SI (producing IL-3 and 
KL) murine fibroblasts in MyeloCult H5100 medium (StemCell Technologies) 
supplemented with 10µM hydrocortisone. The long-term cultures are then incubated for 5 
weeks with weekly half-medium changes. Adherent and floating cells will be harvested 
and 50x103 cells plated in colony-forming assays with MethoCult H4435 (containing 
rhKL, rhG-CSF, rhGM-CSF, rhIL-3, rhIL-6, rhEpo) semisolid medium (StemCell 
Technologies). The colonies, derived from long-term culture-initiating cells (LTC-ICs), 
will be scored after an additional 14 days in culture25,26. 
 
Colony-forming cell and self-renewal assay (CFC/replating assays). 
Methylcellulose colony formation assays will be carried out by plating 103 CD34+/CD38- cell 
sorted from CML patient samples in 0.9% MethoCult H4435 (containing rhKL, rhG-
CSF, rhGM-CSF, rhIL-3, rhIL-6, rhEpo)  (Stem Cell Technologies). Colonies will be 
scored after 14 days in culture. To assess self-renewal individual colonies will be plucked 
in 50µl of IMDM/2%FBS, mixed with 150µl of Methocult and replated in 96-well plates 
and analyzed 14 days later. Single wells containing at least one secondary colony will be 
scored as positive for self-renewal as previously described27. 
 
Shipping Instructions:   
Danilo Perrotti, M.D., Ph.D 
Professor, Dept. of Medicine 

 
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Director Basic Hematologic Research 
Greenebaum Cancer Center 
University of Maryland 
655 W. Baltimore Streer, Room 8-045 
Baltimore, MD 21201 
 
 

 
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Appendix B. Pharmacokinetic (PK) Studies for Ruxolitinib Plasma Level Determination 
 
- 
PK Blood Processing, Storage, and Shipment 
 
1. Collect PK blood samples to analyze ruxolitinib plasma concentrations or potentially, 
metabolites of ruxolitinib using 4 mL EDTA(lavender top) Vacutainer tubes at the time points 
indicated in the protocol. 
2. After obtaining the PK blood sample, gently mix evacuated-collection tube thoroughly by 
slowly inverting the collection tube several times. 
3. Place the collection tube in an ice/water bath. 
4. Within 45 min of sample collection, process collection tubes in a refrigerated centrifuge set at 
approximately 2000 x g for 15 minutes at approximately 5 oC. 
5. Transfer plasma into two aliquots (Aliquots A and B) of approximately equal volume, using 
standard laboratory technique, into 2 appropriately-labeled storage tubes. 
6. Label each tube with the patient’s study registration number, the study I.D., cycle, dose 
and the date and time the sample was drawn. 
7. Ship the samples to the laboratory address listed below with a 2 day supply of dry ice. 
Samples must be stored in a freezer set to maintain a temperature of -20°C to -80°C until ready 
for shipment.  
Note: Samples may be batched but they should only be shipped on Monday through Wednesday. 
 
 
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PK Sample Shipping Instructions: 
 
Ship the plasma samples with a 2-day supply of dry ice. On the day of shipment, notify Incyte of 
the pending shipment by email, and include the protocol number (I-RUX-11-09), sample type 
(e.g., plasma), number of samples, and tracking information. 
 
Tom Emm 
Director, Drug Metabolism and Biopharmaceutics 
Incyte Corporation 
Route 141 and Henry Clay Road 
Building E400 
Wilmington, DE 19880 
 
Phone: (302) 498-6775 
Fax: (302) 425-2759 
E-mail: temm@incyte.com