A Pilot Study to Determine the Effects of the Bruton's Tyrosine Kinase (Btk) Inhibitor PCI-32765 on Leukemia Cell Kinetics and Trafficking, Using Heavy Water Labeling in Subjects With Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL)
Phase: PHASE1
Conditions: Leukemia
Sponsor: M.D. Anderson Cancer Center

2012-0086
09-29-2014
Page 1
Protocol Page
A Pilot Study to determine the effects of the Bruton’s tyrosine kinase (Btk)
inhibitor PCI-32765 on leukemia cell kinetics and trafficking, using Heavy Water 
Labeling in subjects with Chronic Lymphocytic Leukemia (CLL) and Small 
Lymphocytic Lymphoma (SLL).
2012-0086
Core Protocol Information
Short Title 
Pilot Study to determine effects of the Btk inhibitor PCI-32765 on leukemia cell kinetics and 
trafficking, using Heavy Water Labeling in subjecfts with CLL and SLL
Study Chair: 
Jan A. Burger
Additional Contact:
Jeannice Y. Theriot
Sarah K. Renner
Leukemia Protocol Review Group
Department:
Leukemia
Phone:
713-792-1865
Unit:
428
Full Title: 
A Pilot Study to determine the effects of the Bruton’s tyrosine kinase (Btk)
inhibitor PCI-32765 on leukemia cell kinetics and trafficking, using Heavy Water 
Labeling in subjects with Chronic Lymphocytic Leukemia (CLL) and Small 
Lymphocytic Lymphoma (SLL).
Protocol Type:
Standard Protocol
Protocol Phase:
N/A
Version Status:
Terminated 08/09/2018
Version:
13
Submitted by:
Jeannice Y. Theriot--2/22/2018 9:46:00 AM
OPR Action:
Accepted by:  Melinda E. Gordon -- 2/28/2018 12:48:05 PM
Which Committee will review this protocol?
 The Clinical Research Committee - (CRC)

2012-0086
09-29-2014
Page 2
Protocol Body
 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
1 
 
 
 
 
A Pilot Study to determine the effects of the Bruton’s tyrosine kinase (Btk) 
inhibitor PCI-32765 on leukemia cell kinetics and trafficking, using Heavy Water  
Labeling in subjects with Chronic Lymphocytic Leukemia (CLL) and Small  
Lymphocytic Lymphoma (SLL). 
 
 
 
 
 
 
 
 
 
 
Principal Investigator at The University of Texas MD Anderson Cancer Center 
Jan A. Burger, M.D. Ph.D. 
Department of Leukemia, Unit 428 
The University of Texas MD Anderson Cancer Center 
PO Box 301402, Houston, TX 77230-1402, USA 
Phone (713) 563-1487 or (713) 792-1865 
FAX (713) 794-4297 
e-mail: jaburger@mdanderson.org 
 
 
 
 
 
Principal Investigator at North Shore - Long Island Jewish Health System 
Nicholas Chiorazzi, M.D.  
The Feinstein Institute for Medical Research  
North Shore - LIJ Health System  
Professor of Cell Biology and of Medicine  
Albert Einstein College of Medicine  
350 Community Drive  
Manhasset, NY 11030  
Phone: 516-562-1090  
Fax:     516-562-1011  
e-mail: nchizzi@nshs.edu  
 
 
 
 
 
 
 
 
 
 

 
 
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Protocol # 2012-0086 
 
 
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1.0 
Introduction 
PCI-32765 is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo [3,4-d]pyrimidin-1-
yl]-1piperidinyl]-2-propen-1-one, CAS Registry Number 936563-96-1.  PCI-32765 is a 
first-in-class selective, irreversible small molecule inhibitor of Bruton’s tyrosine kinase 
(Btk) currently under investigation in B-cell malignancies.  When formulated, the 
investigational product is PCI-32765, a hard gelatin capsule  
 
B cells are lymphocytes with multiple functions in the immune response, including 
antigen presentation, antibody production, and cytokine release.  B cells express cell 
surface immunoglobulin’s (sIgs) that comprise part of the B-cell antigen receptor (BCR); 
the sIgs are the antigen-capturing portion of the BCR, and BCR-associated molecules 
(e.g., CD79a and CD79b) are the units that activate a B cell after binding to antigen.  
Antigen binding induces receptor aggregation and the clustering and activation of 
multiple tyrosine kinases, which in turn activate further downstream signaling pathways.  
The process of B-cell maturation, including Ig chain rearrangement and somatic 
mutation, is tightly regulated, and it is thought that B-cell lymphomas and CLL result 
from mutations acquired during normal B-cell development.  Several lines of evidence 
suggest that signaling through the BCR is necessary to sustain the viability of B-cell 
malignancies.  First, expression of a functional BCR is maintained throughout lymphoma 
progression, even when the non-expressed immunoglobulin heavy chain (IGH) is 
involved in oncogenic translocations and despite prolonged treatment of tumor cells with 
anti-idiotype therapies.  In addition, selective knockdown of BCR components by RNA 
interference results in apoptosis in multiple B-cell lymphoma cell lines.  Recent studies 
indicate that some large B-cell lymphomas have low level tonic activation of kinases 
downstream of the BCR and that inhibition of this tonic signaling can induce apoptosis.  
Primary follicular lymphoma cells have also been found to maintain enhanced signaling 
from the BCR compared with normal B cells.  Finally, at least 2 drugs that target BCR 
signaling (the Syk inhibitor, R788, and the PKCß inhibitor, enzastaurin) have shown 
evidence of anti-tumor effects in human B-cell lymphomas.  
 
The role of Btk in BCR signal transduction is demonstrated by the human genetic 
immunodeficiency disease, X-linked agammaglobulinemia (XLA), and the mouse genetic 
immunodeficiency disease, X-linked immunodeficiency (xid), both caused by a mutation 
in the Btk gene.  These genetic diseases are characterized by reduced BCR signaling and 
a failure to generate mature B cells.  The Btk protein is expressed in most hematopoietic 
cells with the exception of T cells and natural killer (NK) cells, but the selective effect of 
Btk mutations suggests that its primary functional role is in antigen receptor signaling in 
B cells.  Btk is activated by the upstream Src-family kinases Blk, Lyn and Fyn, and Btk 
in turn phosphorylates and activates phospholipase-C (PLC), leading to Ca++ 
mobilization and activation of NFκB and MAP kinase pathways.  Given the central role 
of Btk in BCR signaling and the importance of BCR signaling in lymphoma, inhibition of 
Btk by PCI-32765 may be an effective therapeutic strategy in B-cell malignancies. 
 
 

 
 
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Protocol # 2012-0086 
 
 
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2.0 
Background 
2.1 
Chronic Lymphocytic Leukemia (CLL)  
CLL is the most common form of adult leukemia in the United States.  It has been 
estimated that there are approximately 150,000 individuals living with CLL in the United 
States.  The use of chemoimmunotherapy as initial therapy is associated with high 
response rates, but this therapy is less effective and displays more side effects in elderly 
subjects.  Once disease relapse has occurred after chemoimmunotherapy, second line 
treatment is less effective and response durations are shorter.  An alternative to repeating 
chemoimmunotherapy is to use the monoclonal antibody (mAb) alemtuzumab.  
Alemtuzumab can induce a therapeutic response in up to 30% of subjects, but is 
associated with significant immunosuppression and infections; also, it lacks activity in 
subjects with bulky lymphadenopathy (larger than 5 centimeters;1.  Combinations of 
purine-analogue based regimens have limited activity, and once subjects are fludarabine 
refractory their median survival is <12 months based on published experience2.  
 
Kinase inhibitors affecting signaling of the BCR, such as PCI-32765, are an alternative, 
targeted therapeutic approach for relapsed and elderly CLL subjects, based on promising 
results in early-phase clinical trials and lack of toxicities.  Characteristically, treatment 
with PCI-32765 induces a rapid reduction in lymphadenopathy, which is accompanied by 
a transient lymphocytosis; the latter is thought to be due to re-distribution of CLL cells 
from tissues into the peripheral blood.  Then, inhibition of BCR-related survival signaling 
by PCI-32765 causes “starvation” and a slow-onset, continuous reduction of the leukemia 
cell burden over time.  These mechanisms of action have not been formally demonstrated 
in vivo.  Labeling of the DNA of newly-born CLL cells with deuterium (2H), 
administered in the form of 2H2O prior to therapy with PCI-32765, will allow us to 
determine the effects of PCI-32765 on leukemia cell mobilization from tissues into the 
peripheral blood, and on leukemia cell kinetics (proliferation, survival, and cell death).  
 
This trial will address the overarching question how much of the anti-leukemia activity of 
PCI-32765 is due to re-distribution of CLL cells from the tissues into the blood versus 
direct effects of  PCI-32765 on CLL cell proliferation and cell death by analysis of 
effects of PCI-32765 on CLL cell tissue redistribution and birth rates. The expected result 
is that PCI-32765 affects both, tissue redistribution and birth rates of CLL cells, based on 
pre-clinical work3 and the more mature clinical data with single-agent PCI-327654. These 
information are important not only for better understanding of the mechanism of action of 
PCI-32765, but potentially also for the further development and optimal use of this drug 
in CLL patients. For example, if PCI-32765 would be found to mainly cause CLL cell 
redistribution, without effects on birth rates of CLL cells, one could argue that the drug 
should preferentially be developed in combination with a cytotoxic agent.  
 
2.2 
Heavy Water Labeling in subjects with Chronic Lymphocytic Leukemia (CLL).  
A fundamental disorder in cancer is altered cell kinetics (imbalance in cell proliferation 
and/or death).  The ideal biomarker for cancer would therefore measure these cellular 
kinetic events directly.  CLL is an excellent clinical cancer model for applying a kinetic 
biomarker, by virtue of its accessibility (being a liquid tumor) and its relatively well-
established kinetic basis.  The laboratory of Dr. Marc Hellerstein at the University of 

 
 
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California has developed, patented, and extensively used a direct kinetic biomarker of 
cell proliferation and death in humans.  The technique is based on stable (non-
radioactive) isotope incorporation (e.g., deuterium, 2H) into cellular DNA at the time of 
cell division followed by gas chromatography/mass spectrometry (GC/MS) analysis of 
deuterated deoxyadenosine.  This technology is exclusively licensed to KineMed, Inc., a 
company formed to commercialize kinetic measurements in medical diagnostics and drug 
development.  Previously our understanding of the kinetics of cells in vivo was limited.  
Indeed, most contemporary data that characterize the proliferation rates of cells are 
derived from ex vivo, static measurements using toxic labels, such as 3H-thymidine or 
bromodeoxyuridine (radioactive and mutagenic, respectively;5,6.  These approaches are 
limited not only by inherent toxicity, but also because they have low reproducibility, are 
non-quantitative, and suffer from other technical limitations.  Most importantly, these 
techniques cannot be used safely in humans in vivo.  Therefore, the safe and non-toxic 
2H2O method for accurately measuring cell proliferation and turnover in CLL in vivo 
could prove to be of significant prognostic utility.  After 2H2O is orally ingested, 2H is 
incorporated into all hydrogen-containing molecules that are being synthesized in vivo.  
For our purposes, the deoxyribose (dR) moiety of purine deoxyribonucleotides in DNA 
of dividing cells is a valuable marker.  Blood or bone marrow is obtained at appropriate 
time points, during and after the 2H2O consumption/labeling period, CLL cells are 
isolated, DNA extracted, and the amount of 2H incorporated into deoxyadenosine of 
DNA is quantified by GC/MS, allowing for a measure of cellular proliferation.  2H is not 
radioactive, and there are no known major risks of heavy water ingestion at the doses that 
we will administer 7.  Transient dizziness/vertigo can be experienced in some subjects 
(~15%).  This technique has been used to measure cellular proliferation in both humans 
and animals.  Some examples in humans follow.  
 
2.3 
Cell Division Studies in Humans Using Heavy Water Labeling 
The heavy water method has been applied to measure the kinetics of human T cells in 
normal and HIV infected individuals, showing a significantly increased T-cell turnover 
rate in untreated HIV+ subjects that improves with treatment (5).  This technique has also 
been used to assess breast epithelial cell proliferation in women from samples taken with 
a core needle biopsy; it was found that pre-menopausal woman had a significantly 
increased cell turnover compared to postmenopausal women. 
 

 
 
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Figure 3. (A) Isotopic labeling of three human serum 
albumin peptides in vivo over time showing the relative 
increase in heavier mass isotopomers (M2 and above) 
and the decrease in light mass isotopomers (M0, M1).  
(B) The fractional synthetic rate of the same peptides 
over time.  (C) Representative mass isotopomer 
distribution of an unlabeled and labeled peptide, 
showing the shift in centroid mass. 
Dynamic Proteomics  
KineMed has developed a new 
technological 
platform 
based 
on 
quantitation of turnover rates of 
multiple proteins simultaneously8. 
This powerful “dynamic proteomics” 
technique can be used in living 
animals and humans8-10 (Figure. 3). 
The kinetics of hundreds of proteins 
are measured concurrently, using 
stable isotope labeling (e.g., with 
heavy water) in vivo followed by 
analysis of trypsin-derived peptides 
by 
liquid 
chromatographytandemmass 
spectrometry 
(LC/MS-MS). 
The 
perturbation in mass isotopomer 
patterns in peptides reveals the 
synthesis and/or removal rates of 
their parent proteins 
9,10. In this 
manner, KineMed recently measured 
the proteome dynamic signature of a 
variety of cell types and conditions in 
mice, including liver mitochondrial 
proteins after long-term CR, muscle 
proteins after anabolic and catabolic 
interventions, 
hippocampal 
progenitor cell proteins in response to 
neurogenic agents and, in humans 
plasma proteins in healthy controls 
and CSF proteins in Parkinson’s 
disease patients. The “unsupervised” 
or non-hypothesis-driven nature of 
the proteome dynamics approach 
allows 
discovery 
of 
previously 
unsuspected molecular targets, while the functional interpretability of many protein 
dynamic processes allows subsequent focus on specific targets of interventions.  This 
new combination of proteomics with kinetics is unique to KineMed and has the power to 
see much deeper into the biology of the disease than a single readout such as collagen 
turnover. 
 

 
 
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Figure 4: Dynamic proteomic analysis of slow vs. fast B-CLL. 440 
protein synthesis rates measured in fast and slow subject. Up-
regulated proteins synthesis rates in the fast subjects in gene ontology 
groups corresponding to cell proliferation and cell cycle. 
KineMed has developed a 
technique for measuring cell 
proliferation rates 
in 
vivo, 
based on 2H2O labeling and 
mass spectrometric analysis, 
which has been applied to the 
measurement 
of 
the 
proliferation of numerous cell 
types in experimental animals 
and humans (original KineMed 
CLL 
cell 
proliferation 
biomarker)11. Cell proliferation 
is commonly measured via 
[3H]thymidine 
or 
BrdU 
techniques, which are known to have toxic effects in animals, while 2H2O can be safely 
administered for long periods of time in drinking water. Currently, our published methods 
using 2H2O labeling are the only techniques for measurement of global protein turnover 
(dynamic proteomics) in rodents or humans. Our laboratory has successfully combined 
2H2O labeling with LC-MS/MS analysis of tryptic peptides to quantify the turnover of 
cellular proteins in vivo8.  These methods are well suited for the measurement of cell and 
protein proliferation in slow turnover tissues because 2H2O can be conveniently 
administered for long periods of time in drinking water, combined with the high 
sensitivity of mass spectrometric analysis of tryptic peptides12. Using 2H2O labeling in 
combination with cell proliferation analysis and dynamic proteomic techniques, KineMed 
proposed measuring the in vivo turnover rate of the B-CLL proteome before and after 
Pharmacyclixs treatment. 
 
 KineMed previously measured CD19+/CD5+ isolated cell pellets from subjects that 
were recruited and labeled as part of the KineMed and CRC cell proliferation biomarker 
clinical trial (Figure 4 and 5) and measured protein and DNA on selected subjects. They 
feel that a dynamic proteomic analysis of the 
Pharmacyclixs cells would provide additional 
information. Since protein turnover occurs 
more rapidly than cell division they may be 
able to label for less time than needed for the 
cell proliferation biomarker.  KineMed  will use 
the dynamic proteomics techniques they 
recently developed9 to fully define the kinetics 
of the CLL B cell. Specifically, they plan to 
measure the turnover rates of a large number of 
proteins and correlate them against the DNA 
turnover rates to generate a complete kinetic 
profile of the CLL B-cell. 
 
 
 
Figure 5: Network analysis of protein 
synthesis rates from fast vs. slow 
subjects 
A)  Ingenuity analysis of transcription 
factors responsible for protein up-
regulation observed in the fast CLL 
subject 
A

 
 
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2.4 
Results in CLL 
Nicholas Chiorazzi and his colleagues at The Feinstein Institute for Medical Research of 
the North Shore - LIJ Health System have used the technique to successfully determine 
the kinetics of CLL cells in subjects 11.  Nineteen subjects imbibed heavy water for 12 
weeks, and blood was collected at multiple time points during and after heavy water 
ingestion.  CLL cells were isolated and analyzed for GC/MS analysis.  The kinetic studies 
revealed significantly higher levels of proliferation than expected, with subject to subject 
variation in CLL birth rates ranging from 0.1% – 1.7%/day.  Those subjects with a more 
rapid rate (defined as > 0.35%/day) were more likely to have progressive/active disease 
(8/10) than subjects with lower birth rates (< 0.35%/day) (1/9), as defined by the NCI- 
Working Group criteria 13. 
 
2.5 
PCI-32765 
Antigenic stimulation through the BCR promotes expansion of CLL and other malignant 
B cells 14,15.  Btk, a key component of BCR signaling, can be blocked by PCI-32765, a 
small-molecule Btk inhibitor that displayed activity in subjects with CLL and other B cell 
malignancies in the first clinical trial 16.  PCI-32765 is a potent (IC50, 0.5nM) and 
selective Inhibitor of Btk that binds covalently to a cysteine residue (Cys-481) in the 
active site of Btk, leading to irreversible inhibition of its enzymatic activity 17.  Once 
daily oral dosing results in 24-hour sustained target occupancy.  In vitro studies 
demonstrated that inhibition of Btk in CLL cells blocks BCR-related survival signals, 
thereby leading to apoptosis 18.  Additionally, Dr. Jan Burger’s laboratory at MD 
Anderson Cancer Center has demonstrated that inhibition of Btk with PCI-32765 
markedly decreases CLL cell responses to chemotactic factors elaborated by stromal 
cells, such as CXCL12 and CXCL13, resulting in diminished CLL cell migration and 
homing capabilities 18.  A recent analysis of data from 38 CLL subjects involved in an 
ongoing Phase Ib/II trial of single-agent PCI-32765 in CLL or SLL included subjects 
with relapsed or refractory disease following at least 2 treatment regimens, and a cohort 
of subjects over 65 years old with treatment naïve disease 16.  When measured clinically 
and by CT scan every 2 months, subjects treated with PCI-32765 typically had an 
immediate and marked nodal response; 87% of subjects with evaluable nodal disease 
achieved a nodal response.  There have been no cases of primary lymph node 
progression.  For the Phase Ia trial with a median follow-up of 8 months, there were 13 
evaluable subjects with 1 CR (8%), 8 PRs (62%) and 2 subjects with a nodal response 
with lymphocytosis (15%).  Two additional subjects had stable disease.  There have been 
only 3 subjects with disease progression, and an additional 3 subjects discontinued 
treatment for reasons other than progressive disease.  Of 32 evaluable subjects in the 
Phase Ib study (with less than 2 months median follow-up), there were 8 PRs (25%) and 
an additional 17 subjects with a nodal response with lymphocytosis.  An additional two 
subjects have stable disease.  As such, the overall response rate (ORR) from the Phase 
Ib/II trial experience at this point is 38% (1 CR, 16 PRs in 45 subjects).  Ninety-four (94) 
percent of subjects experienced an adverse event (AE); 38% subjects experienced an 
adverse event grade 3 or greater in severity (nausea, vomiting, fatigue, diarrhea, 
headache, rash).  The incidence of ≥ grade 3 AEs believed to be related to PCI-32765 was 
17%.  Fourteen subjects experienced a serious adverse event (SAE), with “serious” 
defined as requirement for hospitalization.  Seven SAEs were considered related to study 

 
 
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drug by the investigator.  These were one case of viral lymphadenitis, one case of a 
subdural hematoma, one case of viral infection (follow-up reported as unrelated), one 
case of decreased appetite, dehydration, and asthenia, one case of sepsis, and one case of 
headache and dizziness (follow-up reported as unrelated viral vestibular neuritis) and one 
case of hemorrhagic colitis 16.  Two subjects in this pooled analysis died from progressive 
disease.  Diarrhea, nausea, and fatigue have been the most commonly reported AEs; with 
most of these occurring early in treatment (cycles 1 or 2).  Significant myelosuppression 
has been infrequent; with grade 3 or greater thrombocytopenia or neutropenia reported in 
less than 5% of subjects.  Significant hepatic or renal toxicity has been absent. In 
addition, with longer-term follow-up, there has been no evidence of cumulative toxicity, 
either overall or related to specific events.  Because of the controversial role of Btk in 
platelet function and aggregation, subjects with a history of bleeding diathesis or 
coagulopathy, recent surgical procedure, stroke, or hemorrhage, and subjects receiving 
Coumadin will be excluded. 
 
3.0 
Adverse Events  
PCI-32765 Serious Adverse Events 
Allergic reaction:  A dose limiting toxicity (DLT) hypersensitivity reaction occurred in 
the first-in-human study.  Rash and petechiae have been reported in 13% of subjects.  
Most reports of rash and petechiae have been mild to moderate, however the DLT of 
hypersensitivity suggests that the potential exists for a more severe reaction in sensitive 
individuals.  
 
Leukocytosis in CLL/SLL:  An initial leukocytosis is often observed in subjects with 
CLL/SLL treated with PCI-32765; the leukocytosis decreases steadily over time. 
 
*Of note, an initial leukocytosis has been observed in subjects with CLL/SLL treated with 
PCI-32765; the leukocytosis decreases steadily over time.  The leukocytosis is postulated 
to be a pharmacodynamic effect of PCI-32765 causing lymphocytes to migrate to the 
blood from the lymph nodes, as substantial decreases in lymph node size have been 
observed to correlate with the leukocytosis. This phenomenon will be a prime analysis of 
the heavy water approach. 
 
Infections:  Six subjects have had pneumonia as an SAE.  Although pneumonia is a 
common infectious complication in subjects with lymphoid malignancy, a relationship to 
study drug cannot be ruled out at this time.  Two subjects reported Viral Lymphadenitis. 
 
Asthenia: was reported by one subject 
 
Dehydration and Decreased Appetite:  Was reported by one subject. 
 
Subdural Hematoma:  One subject developed a subdural hematoma. 
 
Hemorrhagic Colitis:  Seventy-three year old female developed, grade 3 diarrhea 9 days 
after starting study drug.  The diarrhea progressively worsened and was hospitalized for 
hemorrhagic colitis.  The Investigator assessed the event as possible related to study drug. 

 
 
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Common adverse events:   
Heavy Water: 
2H2O also occurs naturally, and a minor component of the water ingested daily contains 
2H2O.  The only known side effect of 2H2O ingestion, at the amounts to be ingested in 
this study, is a sense of lightheadedness.  Less frequently, vertigo may occur when the 
compound is ingested too rapidly; this lasts until equilibration within the semi-circular 
canals of the ear is reached.  In the unlikely event that dizziness occurs, it usually 
subsides spontaneously within three hours.   
 
PCI-32765: 
So far, the most commonly reported treatment-emergent adverse events considered 
related to PCI-32765 have been diarrhea (29%), fatigue (17%), nausea (11%), vomiting 
(9%), and rash (8%).  Most of these events also have been mild to moderate in severity.  
 
The above is preliminary safety information.  The information has been reviewed, but has 
not been completely monitored; therefore, minor changes to the safety information may 
occur going forward.  Safety information will continue to be collected and evaluated as 
the study continues.  
 
There were seven (7) deaths within 30 days of last dose of PCI 32765 as of 1/4/11.  All 
deaths were due to disease progression and were reported as unrelated to PCI 32765. 
 
Potential for Drug-Drug Interactions  
Because these PCI-32765 IC50 values are markedly higher than the plasma levels of 
unbound PCI-32765 observed for the initial Phase 1 clinical study, inhibition of P-gp 
transport and CYP450 isoenzyme metabolism is not expected to occur in humans.  
 
However, PCI-32765 is extensively metabolized by CYP2D6 and CYP3A4.  Therefore, 
any medications that are strong inhibitors of CYP2D6 and/or CYP3A4 (eg, itraconazole, 
ketoconazole, clarithromycin, and bupropion) should be administered with caution and 
only after consultation with the Medical Monitor. 
 
4.0 
Objectives 
The primary objective is to assess the impact of the Bruton’s tyrosine kinase (Btk) 
inhibitor PCI-32765 on leukemia cell trafficking and death using heavy water (2H2O) 
labeling of newly-born malignant cells in subjects with Chronic Lymphocytic Leukemia 
(CLL) and Small Lymphocytic Lymphoma (SLL).  
 
The secondary objective is to determine the percent of recently born versus older 
leukemia cells mobilized into the blood by PCI-32765 treatment.   
 
The tertiary objective is to compare the kinetic biomarker measurements in CLL subjects 
to other established prognostic tests and response to therapy with PCI-32765. 
 

 
 
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5.0 
Study Design 
This is a one center, open label pilot study to evaluate the impact of the Btk inhibitor 
PCI-32765 on leukemia cell trafficking and death, using heavy water labeling in subjects 
with Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL).  
PCI-32765 will be supplied by Pharmacyclics as capsules containing 140mg of PCI-
32765.  Subject grade 2H2O will be purchased from KineMed, Inc, and KineMed will 
analyze in vivo incorporation of 2H into deoxyadenosine of B lymphocytes by GC/MS.  A 
total of 30 subjects are to be enrolled at MD Anderson Cancer Center.   
 
6.0 
Subject Eligibility 
Inclusion Criteria: 
Subjects will be eligible for inclusion in the study if they meet all of the following 
criteria: 
1) A diagnosis of CLL/ SLL and have not been previously treated.  
2) An indication for treatment by 2008 IWCLL Criteria 
3) Male and female subjects of age > 18 years at the time of signing informed 
consent and requiring treatment within the next 2 to 6 months. 
4) Understand and voluntarily sign an informed consent, and be able to comply with 
study procedures and follow-up examinations. 
5) Platelet counts at study entry must be greater than 50,000/μL and absolute 
neutrophil counts at study entry must be greater than 750/μL 
6) Free of prior malignancies for 3 years with exception of currently treated basal 
cell, squamous cell carcinoma of the skin, or carcinoma “in situ” of the cervix or 
breast.  
7) Subjects must be able to contribute the required amount of blood and/or tissue 
without compromising their well-being or care and must weigh at least 110 
pounds 
8) Adequate renal and hepatic function as indicated by all of the following: total 
bilirubin </=1.5 x institutional Upper Limit of Normal (ULN); AST or ALT 
</=2.5 x ULN; and estimated creatinine clearance (CrCl) of > 30 mL/min, as 
calculated by the Cockroft- Gault equation 
9) Participants must be willing to be contacted again for consideration of additional 
studies in the future, such as a blood draw or another action (e.g., bone marrow 
aspiration and/or biopsy) that would be associated with their standard of care, 
unless they consented to such for research purposes. 
10) An ECOG/WHO performance status of 0-2. 
11) Males and females of child bearing potential must have adequate birth control 
protection while on study and for 30 days after the last dose of study drug. The 
couple will use two forms of birth control for the entire time of the study and 30 
days after finishing study. Conception control should include a hormonal method 
(birth control pill, etc.), and a double-barrier methods (condoms with spermicidal, 
sponge with spermicidal, or diaphragm with spermicidal), or abstinence (not 
having sex) will be practiced. 
12)  Female subjects will need a negative pregnancy screening test if they are of child 
bearing potential. 

 
 
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Exclusion Criteria: 
1) Subjects less than 18 years of age 
2) A lymphocyte doubling time of < 3 months, or other clinical or laboratory signs 
indicating that a treatment delay of 2 months or longer (due to heavy water 
labeling and resting period) would result in a significant progression of the 
disease and be detrimental to the subject, as determined by the treating physician 
3) Any prior treatment for CLL including chemotherapy, chemoimmunotherapy, 
monoclonal antibody therapy, radiotherapy, or high-dose corticosteroid therapy 
(Prednisone > 60 mg daily or equivalent), or immunotherapy prior to enrollment 
or concurrent with this trial. 
4) Concomitant use of agents that have been described to affect the biology and/or 
proliferation rate of CLL cells such as: PDE-inhibitors (e.g., sildenafil, 
theophylline), immunosuppressive agents (e.g., prednisone, cyclosporin-A, 
rapamycin), green tea extract, itraconazole, ketoconazole, clarithromycin, 
bupropion, and Cox-2 inhibitors 
5) Investigational agent received within 30 days prior to the first dose of study drug.  
If received any investigational agent prior to this time point, drug-related 
toxicities must have recovered to Grade 1 or less prior to first dose of study drug. 
6) Systemic fungal, bacterial, viral, or other infection not controlled (defined as 
exhibiting ongoing signs/symptoms related to the infection and without 
improvement, despite appropriate antibiotics or other treatment). 
7) Subjects with uncontrolled autoimmune hemolytic anemia (AIHA) or 
autoimmune thrombocytopenia (ITP) 
8) Any other severe concurrent disease, or have a history of serious organ 
dysfunction or disease involving the heart, kidney, liver or other organ system that 
may place the subject at undue risk to undergo therapy with PCI-32765. 
9) Any serious medical condition, laboratory abnormality, or psychiatric illness that 
places the subject at unacceptable risk if he/she were to participate in the study. 
10) History of intracranial hemorrhage or stroke within 6 months prior to the study  
11) Evidence of bleeding diathesis or coagulopathy 
12) Major surgical procedure, open biopsy, or significant traumatic injury, within 28 
days prior to Day 1, anticipation of need for major surgical procedure during the 
course of the study.  (Minor surgical procedures, fine needle aspirations or core 
biopsies within 7 days prior to Day 1.  Bone marrow aspiration +/- biopsy is 
allowed). 
13) Serious, non-healing wound, ulcer, or bone fracture. 
14) Subjects receiving anticoagulation (for example heparin, Coumadin, low-
molecular-weight heparin (LMWH, such as Lovenox), and anti-platelet drugs 
(except for low-dose aspirin) will be ineligible to participate in this study. 
Subjects who recently received drugs for anticoagulation must be off those 
medications for at least 7 days prior to start of the study. 
15) Subjects who are known to be anemic, with hemoglobin <8.0g/dl. 
16) Weight less than 110 pounds 
17) PCI-32765 is contraindicated in subjects with clinically significant 
hypersensitivity to any of the compound’s structural components.   

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
12 
 
18) Subjects who are known to be infected with HIV, or have signs of active Hepatitis 
B or Hepatitis C  
19) Biliary obstruction, acute hepatitis, severe liver failure, or severely impaired renal 
function 
 
7.0 
Pretreatment evaluation 
Pretreatment evaluation will include a physical examination including vital signs, 
ECOG/WHO performance status, height and weight, and recording of concurrent 
medications, and will be done within 30 days prior to day 1  (See Appendix A). 
 
Clinical laboratory evaluation will involve serum chemistries, including sodium, 
potassium, calcium, magnesium, phosphorus, BUN, creatinine, glucose, albumin, total 
protein, alkaline phosphatase, total bilirubin, ALT, uric acid, andBeta-2-microglobulin 
levels Hematology studies will include complete blood count (CBC) with differential and 
peripheral blood lymphocyte subset analysis, immunoglobulin levels (within 30 days 
prior to day 1), and coagulation studies (Prothrombin Time/PT; Partial Thromboplastin 
Time/PTT; and International Normalized Ratio/ INR) within 30 days prior to day 1.  
Flow cytometry assessment for peripheral CLL cells (CLL panel) and lymphocyte 
subpopulations (CD3, CD4, CD8, CD19, CD16/56 cells, 30 days prior to day 1). 
 
Bone marrow aspiration and biopsy will be obtained within one month of day 1 of the 
study, and again after 3, 6, and 12 cycles (+/- 1 month), only bone marrow aspirations (no 
biopsy) will be done during follow-up evaluations after 3, 6, and 12 cycles) after 
initiating PCI-32765 therapy.  The screening bone marrow will be evaluated by flow 
cytometry for clonality and for IGHV mutation studies (unless known), CD38 expression, 
ZAP-70 expression, and cytogenetic and genomic abnormalities by FISH. Follow-up 
bone marrow tests after 3, 6, and 12 cycles (+/- 1 month) will evaluate morphology (bone 
marrow lymphocyte quantification), and quantification of CLL cells by flow cytometry, 
including minimal residual disease assessment by flow cytometry. Electrocardiogram 
(within one month from registration) will be required.  We will not require additional 
ECG monitoring during this study.  As of April 6, 2012, 408 subjects have been treated 
with ibrutinib in 8 clinical studies.  The cardiovascular safety of ibrutinib was evaluated 
by monitoring of adverse events in all studies; in addition, a formal ECG monitoring was 
performed in 2 studies (PCYC-04753 & PCYC-1102-CA).  To date there is no evidence 
of ECG morphological changes or prolongation of QTc in patients treated with 
ibrutinib.  The most common cardiovascular adverse events reported across the studies 
were atrial fibrillation (n=17), tachycardia (n=7), sinus tachycardia (n=4) and sinus 
bradycardia (n=3).  Events of Grade 3 or greater severity included only atrial fibrillation 
(n=6), supraventricular tachycardia (n=2) and tachycardia (n=1).  These findings are 
consistent with expectations in elderly population of subjects many of whom have known 
cardiovascular disease at baseline. Because of these data we will not request follow-up 
ECGs.  Subjects also will undergo CT scans of the chest, abdomen, and pelvis after 3 or 6 
cycles (+/- 1 month) and after 12 cycles (+/- 1 month) of PCI-32765 therapy. 
Anti-coagulated blood sample (50ml) and coagulated blood sample (10ml) for baseline 
2H2O studies on the day of study initiation (Day 1). 
 

 
 
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8.0 
Treatment Plan 
8.1 
Heavy Water Protocol: Labeling phase. 
After enrollment and a baseline blood draw (60ml), subjects will start to consume heavy 
water (2H2O).  Subjects will be given 50 mL of 70% 2H2O 3 times a day for the first 5 
days followed by 60ml daily for a total of 4 weeks (labeling phase).  Lightheadedness or 
dizziness may occur for some subjects (~15%) with the initial doses; therefore subjects 
will be given their first dose in clinic and asked to avoid driving or performing potentially 
hazardous activities for one hour.  If such symptoms occur, they usually abate within 2-3 
hours.  Subjects will then be given individual doses of 2H2O to consume at home; after 
the 5-day loading period, a 60 mL maintenance dose of 2H2O will be drunk at bedtime to 
obviate any problems with dizziness. Compliance with 2H2O ingestion will be 
documented using a heavy water ingestion diary (intake logs). Unused 2H2O will be 
returned for disposal. 
 
Subjects will be seen at day 1 (+/- 3 days), day 14 (+/- 3 days), and day 28 (+/- 3 days) 
after beginning 2H2O ingestion for follow-up visits with clinical and laboratory 
assessment, which will include determining if there have been any problems with 2H2O 
ingestion, analysis and discussion of intake logs, and blood sampling (60ml) for CLL cell 
kinetic measurements (heavy water testing).   
 
8.2 
Heavy Water Protocol: Resting phase. 
At the end of the 4th week, subjects will stop drinking 2H2O (washout phase) and be 
followed for 6-12 weeks until beginning treatment with PCI-32765.  During the period 
until initiation of therapy, subjects will be seen every 2 weeks (+/- 3 days) for follow-up 
visits with clinical and laboratory assessment. Blood will be drawn at the same time 
every 2 weeks (+/- 3 days), to determine a disappearance rate (death plus re-distribution 
into solid tissue) of CLL cells containing 2H-labeled DNA.  Only those subjects that have 
not imbibed 2H2O for at least 6 weeks from the end of the labeling period until starting on 
treatment with PCI-32765 or those patients who have not imbibed heavy water for less 
than 6 weeks and have received study PI approval will be included in further 2H2O 
kinetic studies, although they will receive PCI-32765 as a part of the response to therapy 
studies. After start of treatment with PCI-32765, there will be blood sampling (60ml) for 
CLL cell kinetic measurements (heavy water testing) on days 1 (+/- 3 days), 3 (+/- 1 day), 
and then at week 2 (+/- 3 days) and every 2 weeks (+/- 3 days) thereafter for a period of 
at least equal in duration to that of the initial 2H2O washout phase (6 to 12 weeks). 
Patients will continue to return to clinic each month for blood drawing (60ml). Data 
obtained from these blood samples will permit a determination of CLL birth rate as well 
as a slope of labeled cell emergence from solid tissue into the peripheral blood.     
 
8.3 
Treatment with PCI-32765 
PCI-32765 will be administered with 8 ounces (~240mL) of water at a dose of 420 mg (3 
x 140mg capsules) orally once daily and will be continued daily. A PCI-32765 dose of 
420 MG PO once daily is based on the experience in Phase 1 and Phase 2 trials which 
established this dose and schedule to be adequate to induce BTK target occupancy (see 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
14 
 
Advani et al, JCO 201219). 420 mg is the dose used in all of the CLL/SLL trials to 
date. Each dose of PCI-32765 should be taken at least 30 minutes before eating and at 
least 2 hours after a meal at approximately the same time each day. Subjects will be 
instructed to avoid grapefruit juice due to CYP450 3A4 inhibition. Compliance with PCI-
32765 ingestion will be documented using a diary. Treatment duration will be 12 cycles, 
with each cycle consisting of 28 days.  Response will be evaluated after 3, 6 and 12 
cycles.  It will be possible for subjects to continue taking the drug beyond 12 cycles if 
there is a significant benefit such as an ongoing PR or CR. Unused PCI-32765 will be 
returned for disposal per institutional policy. 
 
8.4 
Special Handling Instructions 
Allopurinol at the dose of 300mg daily will be given during the first one to two weeks of 
treatment as standard tumor lysis prophylaxis. 
 
8.5 
Concomitant Medication 
Subjects receiving anticoagulation (for example heparin, Coumadin, low-molecular-
weight heparin (LMWH, such as Lovenox), and anti-platelet drugs (except for low-dose 
aspirin) will be ineligible to participate in this study.  Subjects who recently received 
drugs for anticoagulation must be off those medications for at least 7 days prior to start of 
the study. 
 
8.6 
Guideline for Use of CYP Inhibiting Drugs 
 
PCI-32765 is metabolized by CYP3A4/5 and CYP2D6.  Alternatives to strong 
CYP3A4/5 inhibitors (such as clarithromycin, ketoconazole, itraconazole, nefazodone, 
and ritonavir) and strong CYP2D6 inhibitors (such as bupropion, fluxetine, paroxetine, 
and quinidine) should be sought if possible. 
 
If no alternative treatment is available, strong CYP3A4/5 and CYP2D6 inhibitors should 
be used with caution; patients should be closely monitored for potential toxicities with 
temporary interruption of PCI-32765 as appropriate.  
  
A comprehensive list of cytochrome P450 isoenzymes and CYP3A4/5 and CYP2D6 
inhibitors, inducers, and substrates can be found at http://medicine.iupui.edu/flockhart.  
This website is continually revised and should be checked frequently for updates. 
 
8.7 
Concomitant Use of QT Prolonging Agents 
 
During the course of study drug treatment medications known to cause Torsades des 
Pointes should be avoided.  Medications known to cause QT prolongation may be used 
with caution. 
 
 
8.8 
Concomitant Use of Antiplatelet Agents and Anticoagulants 
 

 
 
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Protocol # 2012-0086 
 
 
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Laboratory studies have shown that, in vitro, PCI-32765 can prevent platelets from 
aggregating normally; the clinical significance of this finding is unknown at this time.  
While serious bleeding has been uncommon in patients treated to date, it is possible that 
treatment with the study drug could increase the risk of bruising or bleeding, particularly 
in subjects receiving other antiplatelet agents or anticoagulants. 
Subjects receiving antiplatelet agents in conjunction with PCI-32765 should be observed 
closely for any signs of bleeding or bruising, and PCI-32765 should be withheld in the 
event of any bleeding events.  
Subjects requiring the initiation of anticoagulation with warfarin or related agents during 
the course of the study should have treatment with PCI-32765 held, and PCI-32765 
should not be restarted until subjects are stably anticoagulated.  During the co-
administration of PCI-32765 and anticoagulant therapy, the INR should be monitored 
carefully and subjects should be observed closely for signs and symptoms of bleeding. 
 
 
8.9 
Sample Collections for 2H-DNA-labeled Leukemic Cells during PCI-32765 
Treatment Phase 
After beginning PCI-32765 therapy, subjects will be seen for follow-up visits including 
blood draws (60ml) at day 1 (+/- 3 days), day 3 (+/- 1 day), week 2 (+/- 3 days), and 
every 2 weeks (+/- 3 days) thereafter for a period at least equal in duration to that of the 
initial 2H2O washout phase of 6 to 12 weeks.  At these visits, clinical and laboratory 
assessment, which will include blood sampling (60ml) for 2H2O kinetic measurements, 
will be made.  Subjects will continue to return or be seen by a local physician or clinical 
laboratory each month (+/- 3 days) for an additional 3 months for blood drawing (60ml). 
Data obtained from these blood samples will permit a determination of the slope and rate 
of the appearance of previously labeled CLL cells in the blood, and the slope and rate of 
the disappearance of labeled cells from the blood.  If re-circulation to solid tissues is 
inhibited by PCI-32765 therapy, the disappearance rate will represent primarily the death 
rate of leukemic cells in vivo.  Because during the treatment period, 2H2O will not be 
available to label newly-born cells, any cells containing 2H-labeled DNA will represent 
cells labeled during the initial period of 2H2O consumption and retained in situ.  The 
kinetic measurements of the entire CLL clone will be compared with those of intraclonal 
fractions from the same samples, defined by the reciprocal differences in CXCR4/CD5 
surface membrane densities; this approach is described in the published study by 
Calissano et al. (Mol Med 2011;17: 1374-1382, 2011).  In addition, these fractions will 
be analyzed for: 
1. Phenotype and numerical changes of B and T cells and myeloid cells within 
these fractions, pre- and post-ibrutinib therapy  
2. Gene expression profiles of cells within these fractions, pre- and post-ibrutinib 
therapy 
3. DNA methylation profiles of cells within these fractions, pre- and post-
ibrutinib therapy. 

 
 
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Protocol # 2012-0086 
 
 
16 
 
4. Response to BCR and other signals of cells within these fractions, pre- and 
post-ibrutinib therapy,using western blots on phosphoproteins/ibrutinib 
occupancy 
5. Differences in engraftment and growth capacities of CLL B and T cells in 
NSG mice, unseparated or within these fractions, pre- and post-ibrutinib 
therapy 
 
All data will be correlated with the kinetic data of the clone and its CXCR4/CD5 subsets 
in order to better characterize CLL cell subpopulations that are affected by PCI-32765 
therapy and that account for CLL cell mobilization and/or proliferation. These additional 
analyses will be done at Dr. Chiorazzi’s site, in collaboration with Dr. Burger and his 
colleagues at MD Anderson Cancer Center. 
 
8.10 
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 (see Appendix 
H) 
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 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 month for the first 3 months of starting 
treatment with PCI-32765, and then every 3 cycles for evaluation (every 6 months 
after cycle 36) 
 
9.0 
Special Instructions about Heavy Water Ingestion: 
Related to heavy water 
“Heavy water” is similar to “regular water” in taste, smell, and color.  There are no 
known major harmful effects of heavy water when consumed in the amounts given in this 
study.  Minor side effects include nausea, dizziness, lightheadedness, and vertigo.  
Participants will be advised to avoid performing activities such as driving, operating 
machinery, and exercise that could potentially compromise their safety immediately after 
drinking the heavy water.  They should wait at least 60 minutes before resuming any 
strenuous activities. Unused 2H2O will be returned for disposal. 

 
 
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17 
 
 
Special Instructions Related to contraception    
Heavy Water: 
Even though no pregnant women will be enrolled in this study, deuteriated compounds 
including deuteriated vitamin E, deuteriated nicotine, and deuteriated water have been 
given to pregnant women to study the physiology of pregnancy and no ill effects to 
mother or fetus have been observed.   
 
PCI-32765: 
The potential effects of PCI-32765 on a developing fetus have not been determined in 
animals or humans. 
 
Therefore, if a woman of childbearing potential is enrolled in this study or a man with a 
partner who has childbearing potential, the couple will use two forms of birth control for 
the entire time of the study and 30 days after finishing study.  Conception control should 
include a hormonal method (birth control pill, etc.), and a double-barrier methods 
(condoms with spermicidal, sponge with spermicidal, or diaphragm with spermicidal), or 
abstinence (not having sex) will be practiced. 
 
 
10.0 Evaluation during Study 
• 
Clinic visits, end of treatment visit 
Each clinic visit will include vital signs and a physical exam. Physical examinations 
should include height (screening only) and weight, examination of the skin, eyes (with 
specific questions focused on ocular symptoms), ears, nose, throat, lungs, heart, 
abdomen, extremities, and lymphatic system. The examination should also include 
inquiry of ocular symptoms and subjects should be referred to an ophthalmologist for a 
formal examination if any Grade > 2 symptoms are reported.  After beginning PCI-32765 
therapy, subjects will be seen for follow-up visits including blood draws (60ml) on days 1 
(+/- 3 days), 3 (+/- 1 day), and then at week 2 (+/- 3 days), and every 2 weeks (+/- 3 days) 
thereafter for a period at least equal in duration to that of the initial 2H2O washout phase 
of 6 to 12 weeks.  Subjects will return to clinic or will be seen by a local physician each 
month (+/- 3 days) for an additional 3 months for follow-up visits with clinical and 
laboratory assessment, including vital signs and physical exam, and laboratory workup, 
including hematology and clinical chemistry evaluations and then every 3 months (+/- 3 
days) until the end of 12 cycles.  If subjects stay on the study past 12 cycles, they will 
return for clinic visits for clinical assessment, including vital signs and physical exam, 
and laboratory workup, including hematology and clinical chemistry evaluations, very 3 
months (+/- 1 month) for cycles 13 through 36, and every 6 months (+/- 1 month) 
thereafter. For patients that discontinue therapy with PCI-32765, an end of study visit at 
MD Anderson will be required. Patients will be followed for at least 2 months (+/- 1 
month) after the last dose of PCI-32765 for assessment of adverse events. This will be 
done in a Leukemia Clinic visit that will be scheduled to occur at 1 month (+/- 10 days) 
after the last dose of PCI-32765.   
 

 
 
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18 
 
• Hematology and serum chemistry  
CBCs (white blood cell, hemoglobin, platelets and differential) will be monitored on days 
1, 14, and 28 (+/- 3 days) and then every 4 weeks (+/- 3 days) thereafter.  Serum 
chemistry including basic metabolic profile (sodium, potassium, chloride, CO2, BUN, 
creatinine and glucose) will be monitored once weekly (+/- 3 days) for the first 4 weeks 
and then once every 4 weeks (+/- 3 days) thereafter until the end of cycle 12.  If subjects 
stay in the study past 12 cycles, they will have these laboratory tests (hematology and 
clinical chemistry) done every 3 months (+/- 1 month) for cycles 13 through 36, and 
every 6 months (+/- 1 month) thereafter during their clinic visit.   
 
PT, PTT, and INR will be monitored after cycles 3 and 12 (+/- 1 week) and if subjects 
stay on the study past 12 cycles, once every 12 cycles (+/- 1 month).  
 
• Bone 
marrow 
testing, 
beta-2-microglobulin, 
lymphocyte 
subset, 
immunoglobulin levels 
Bone marrow testing will be done by flow cytometry after 3 (+/- 1 month), 6 (+/- 1 
month), and 12 cycles (+/- 1 month) and if subjects stay in the study past 12 cycles, once 
every 12 cycles (+/- 1 month). Beta-2 microglobulin and immunoglobulin levels will be 
assessed at screening and at the timepoints specified in the study calendar (see calendar 
of events at the end of the protocol). 
 
• CT scans of the chest, abdomen, and pelvis  
These will be performed within 30 days before starting Heavy Water, and then after 3 or 
6 cycles, and after 12 cycles.  All other scans are at the treating physician’s discretion. 
 
• Research Testing as an Optional Procedure 
Patient samples (blood cells, DNA, RNA) will be analyzed by colleagues at the 
Dana-Farber Cancer Institute (DFCI), Boston (Dr. Catherine Wu and associates) 
for gene mutations within the CLL cells in patients responding or not responding 
to therapy with PCI-32765.  These tests will allow us to determine why some 
CLL cells persist during therapy with PCI-32765 and why some patients have 
suboptimal responses or no response to PCI-32765. No additional blood draws 
will be necessary for these tests. 
 
Blood samples will be analyzed by Dr. Richard Eric Davis (Department of 
Lymphoma and Myeloma, MD Anderson) for changes in gene expression in CLL 
cells during therapy with PCI-32765. For these studies, one additional tube of 
blood (approximately 1 tablespoon per time point) will be collected on cycle 1, 
week 1, day 1 (+/- 3 days) before the start of PCI-32765, and day 3 (+/-1 day), 
and on week 4 (+/- 3 days), and week 12 (+/- 3 days), during therapy with PCI-
32765. 
 
Finally, a sputum sample will be collected on day 1 of week 1 (+/- 3 days) during 
follow-up which will serve as a control for the genetic testing of the leukemia cells.  

 
 
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19 
 
Samples collected will be stored at Dr. Burger’s laboratory and then sent to the Dana-
Farber Cancer Institute  
 
• Confidentiality 
All patient-identifying information will be removed from all samples before being 
sent to DFCI and Dr. Richard Davis’ laboratory and will only be available to the 
PI at MD Anderson, Dr. Jan Burger. 
 
11.0 Criteria for Response 
The International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria will 
be used 20.  Responses will be assessed after 3, 6 and 12 cycles. 
 
12.0 Evaluation of Toxicity 
Safety Results Among Treatment-naïve Elderly Patients 
The safety profile of treatment-naïve elderly patients receiving PCI-32765 has been 
consistent with the treated population as a whole, as evidenced by the 31 patients aged 65 
years or older in Study PCYC-1102-CA (data cut 03 July 2012). Diarrhea (58%), nausea 
(50%), and fatigue (35%) have been the most prevalent treatment-emergent AEs. Of the 
31 treatment-naïve CLL patients aged 65 years or older in study PCYC-1102-CA, 42% 
experienced a Grade 3 or 4 AE (32% and 10%, respectively). Three patients had Grade 3 
diarrhea. The only Grade 4 AEs were anemia, thrombocytopenia, and neutropenia (1 
patient each).  Two out of the 31 elderly patients (6.5%) experienced an AE leading to 
study drug discontinuation in Study PCYC-1102-CA. The AEs leading to discontinuation 
were Grade 2 viral infection and Grade 3 fatigue. One patient, a 75-year-old man, was 
hospitalized for symptoms of a viral illness that began 35 days after the start of study 
treatment and resolved during the hospitalization. Study drug was discontinued due to 
non-serious symptoms of night sweats and fever that persisted past drug discontinuation 
and were attributed to CLL. The other patient, a 70-year-old woman with a medical 
history of hypothyroidism and intermittent fatigue, was treated with study drug for 
approximately 4 months and had achieved a partial response with lymphocytosis. Study 
treatment was discontinued due to Grade 3 fatigue. 
 
Of note, serial evaluation of quantitative serum immunoglobulins in both 
relapsed/refractory and treatment-naïve elderly patients revealed a statistically significant 
increase in IgA at 3, 6, and 12 months (p<0.005 at each timepoint, multiplicity not 
adjusted) with no decline in IgG or IgM, indicating that PCI-32765 did not impact normal 
immunoglobulin production. 
 
Assessment of Safety 
Safety assessments will consist of monitoring and recording AEs and SAEs, and should 
be consistent with institutional standards and Good Clinical Practice.   
Adverse Events 
An AE is any untoward medical occurrence in a subject administered a pharmaceutical 
product and which does not necessarily have a causal relationship with this treatment. An 

 
 
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Protocol # 2012-0086 
 
 
20 
 
AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding, for example), symptom, or disease temporally associated with the use 
of an investigational study drug, whether or not considered related to the study drug. 
For the purposes of this clinical study, adverse events include only treatment-emergent 
events which are either new or represent detectable exacerbations of pre-existing 
conditions.  
Adverse events may include, but are not limited to: 
• Subjective or objective symptoms spontaneously offered by the subject and/or 
observed by the investigator or study staff including laboratory abnormalities of 
clinical significance. 
• Any adverse events experienced by the subject through the completion of final 
study procedures.  
• AEs not previously observed in the subject that emerge during the protocol-
specified AE reporting period, including signs or symptoms associated with MCL 
that were not present before the AE reporting period 
• Complications that occur as a result of protocol-mandated interventions (eg, 
invasive procedures such as biopsies) 
The following are NOT considered an adverse event: 
• Pre-existing condition: A pre-existing condition (documented on the medical 
history CRF) is not considered an AE unless the severity, frequency, or character 
of the event worsens during the study period. 
• Preplanned hospitalization: A hospitalization planned before signing the 
informed consent form is not considered an SAE, but rather a therapeutic 
intervention. However, if during the pre-planned hospitalization an event occurs, 
which prolongs the hospitalization or meets any other SAE criteria, the event will 
be considered an SAE. Surgeries or interventions that were under consideration 
but not performed before enrollment in the study will not be considered serious if 
they are performed after enrollment in the study for a condition that has not 
changed from its baseline level.  Hospitalizations for social reasons or due to long 
travel distances are also not SAEs. 
• Diagnostic Testing and Procedures: Testing and procedures should not to be 
reported as adverse events or serious adverse events, but rather the cause for the 
test or procedure should be reported. 
Suspected Adverse Reaction 
A Suspected Adverse Reaction is any adverse event for which there is a “reasonable 
possibility” that the drug caused the adverse event.    

 
 
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Protocol # 2012-0086 
 
 
21 
 
“Reasonable Possibility”, for the purposes of safety reporting, means there is evidence to 
suggest a causal relationship between the drug and the adverse event.  Examples of 
evidence that would suggest a causal relationship between the drug and the adverse event 
are: 
• A single occurrence of an event that is uncommon and known to be strongly 
associated 
with 
drug 
exposure 
(eg, 
angioedema, 
blood 
dyscrasias, 
rhabdomyolysis, hepatic injury, anaphylaxis, and Stevens-Johnson Syndrome).  
• One or more occurrences of an event that is not commonly associated with drug 
exposure, but is otherwise uncommon in the population exposed to the drug (eg, 
include tendon rupture or heart valve lesions in young adults, or intussusception 
in healthy infants).  If the event occurs in association with other factors strongly 
suggesting causation (eg, strong temporal association, event recurs on 
rechallenge), a single case may be sufficiently persuasive; but often, more than 
one occurrence (from one or multiple studies) would be needed before the sponsor 
could make a determination of whether the drug caused the event.  
• An aggregate analysis of specific events that can be anticipated to occur in the 
study population independent of drug exposure.  Such events include known 
consequences of the underlying disease or condition under investigation (eg, 
symptoms or disease progression), or events unlikely to be related to the 
underlying disease or condition under investigation, but commonly occur in the 
study population independent of drug therapy (eg, cardiovascular events in an 
elderly population).  An aggregate analysis (across studies) will identify those 
events that occur more frequently in the drug treatment group than in a concurrent 
or historical control group. 
 Severity 
Definitions found in the Common Terminology Criteria for Adverse Events version 4.0 
(CTCAE v4.0) will be used for grading the severity (intensity) of AEs. The CTCAE v4.0 
displays Grades 1 through 5 with unique clinical descriptions of severity for each 
referenced AE. Should a patient experience any AE not listed in the CTCAE v4.0, the 
following grading system should be used to assess severity: 
• Grade 1 (Mild AE) – experiences which are usually transient, requiring no special 
treatment, and not interfering with the patient’s daily activities 
• Grade 2 (Moderate AE) – experiences which introduce some level of inconvenience 
or concern to the patient, and which may interfere with daily activities, but are usually 
ameliorated by simple therapeutic measures 
• Grade 3 (Severe AE) – experiences which are unacceptable or intolerable, 
significantly interrupt the patient’s usual daily activity, and require systemic drug 
therapy or other treatment 

 
 
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• Grade 4 (Life-threatening or disabling AE) – experiences which cause the patient to 
be in imminent danger of death 
• Grade 5 (Death related to AE) – experiences which result in patient death 
 Causality 
The investigator is to assess the causal relation (ie, whether there is a reasonable 
possibility that the study drug caused the event).  
Unexpected 
An “unexpected” AE is an AE that is not listed in the Investigator Brochure or is not 
listed at the specificity or severity that has been observed.  For example, hepatic necrosis 
would be “unexpected” (by virtue of greater severity) if the Investigator Brochure 
referred only to elevated hepatic enzymes or hepatitis. Similarly, cerebral 
thromboembolism and cerebral vasculitis would be “unexpected” (by virtue of greater 
specificity) if the Investigator Brochure listed only cerebral vascular accidents. 
"Unexpected" also refers to AEs that are mentioned in the Investigator Brochure as 
occurring with a class of drugs or as anticipated from the pharmacological properties of 
the drug, but are not specifically mentioned as occurring with the study drug under 
investigation. 
 
Serious Adverse Event Reporting (SAE) for MD 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 
• 
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). 
 

 
 
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23 
 
• 
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 
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. 
 
 
Protocol-specific dose adjustments  
PCI-32765 dose adjustments for surgical or invasive procedures: 
The following guidance should be applied during the perioperative period for subjects 
who require surgical intervention or an invasive procedure while receiving ibrutinib: 
• 
For any surgery or invasive procedure requiring sutures or staples for closure, 
Ibrutinib should be held at least 7 days prior to the intervention and should be 
held at least 7 days after the procedure, and restarted at the discretion of the 
investigator when the surgical site is reasonably healed without serosanguineous 
drainage or the need for drainage tubes. 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
24 
 
• 
For minor procedures (such as a central line placement, needle biopsy, 
thoracentesis, or paracentesis) ibrutinib should be held for at least 3 days prior to 
the procedure and should not be restarted for at least 3 days after the procedure. 
For bone marrow biopsies that are performed while the subject is on ibrutinib, it is 
not necessary to hold ibrutinib for these procedures. 
• 
For emergency procedures, ibrutinib should be held after the procedure until the 
surgical site is reasonably healed, for at least 7 days after the urgent surgical 
procedure. 
 
PCI-32765 dose adjustments for the following hematologic toxicities:  
• Grade 4 ANC (< 500/µL) for > 7 days  
• Grade 3 or 4 Platelets (< 50,000/µL) in presence of significant bleeding  
• Grade 4 Platelets (< 25,000/µL)  
Gastrointestinal toxicities: 
• Grade 3 or 4 nausea, vomiting or diarrhea if persistent despite optimal antiemetic 
and/or anti-diarrheal therapy) or any other grade 4 or unmanageable grade 3 
toxicities: 
 
Occurrence 
Action 
1st 
Hold PCI-32765 until recovery to Grade ≤ 1 or baseline; may restart at 
original dose level 
2nd 
Hold PCI-32765 until recovery to Grade ≤ 1 or baseline; restart at one dose 
level lower (280 mg daily) 
3rd 
Hold PCI-32765 until recovery to Grade ≤ 1 or baseline; restart at one dose 
level lower (140 mg daily) 
4th 
Discontinue PCI-32765 
 
Events of Special Interest 
Specific adverse events, or groups of adverse events, will be followed as part of standard 
safety monitoring activities by the Sponsor.  These events will be reported to the 
Pharmacyclics within 24 hours of awareness following the procedure described for SAEs 
and will require enhanced data collection.  All Events of Special Interest will be 
submitted without a serious criterion selected if no other serious criterion is met.   
List of Events of Special Interest 
Major Hemorrhage 
Defined as any hemorrhagic event, that is Grade 3 or greater in severity, or that results in 
one of the following: intraocular bleeding causing loss of vision, the need for a 
transfusion of two or more units of red cells or an equivalent amount of whole blood, 
hospitalization or prolongation of hospitalization.  

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
25 
 
Events meeting the definition of major hemorrhage will be captured as an event of special 
interest (see above). 
 
DOCUMENTING AND REPORTING OF ADVERSE AND SERIOUS ADVERSE 
EVENTS 
The investigator is responsible for ensuring that all AEs and SAEs that are observed are 
reported during the study. 
Adverse Event Reporting Period 
The AE reporting period for this study begins when the patient takes the first dose of 
study drug and ends with the safety follow-up visit If an SAE is present at the safety 
follow-up visit, or within 30 days of the last dose of study drug (whichever is later), the 
SAE (and associated AEs and concomitant medications) should be followed to resolution 
or until the Investigator assesses the subject as stable, or the subject is lost to follow-up or 
withdraws consent. Resolution/stable means the subject has returned to baseline state of 
health or the investigator does not expect any further improvement or worsening of the 
event. 
Assessment of Adverse Events 
Investigators will assess the occurrence of AEs and SAEs at all patient evaluation time 
points during the study.  All AEs and SAEs whether volunteered by the patient, 
discovered by study personnel during questioning, or detected through physical 
examination, clinically significant laboratory test, or other means will be recorded in the 
patient’s medical record and on the CRF and, when applicable, on an SAE form. 
Each recorded AE or SAE will be described by its duration (ie, start and end dates), 
severity, regulatory seriousness criteria, if applicable, suspected relationship to the 
investigational product (see following guidance), and any actions taken. 
All SAEs (initial and follow-up information) will be reported by the investigator, or 
designee, to Pharmacyclics, within 24 hours, or the next business day, after discovery of 
the event or information. The sponsor and Pharmacyclics may request follow-up and 
other additional information (eg, hospital admission/discharge notes, and laboratory 
results). 
All deaths should be reported with the primary cause of death as the AE term, as death is 
typically the outcome of the event, not the event itself. The primary cause of death on the 
autopsy report should be the term reported.   
An SAE will qualify for expedited reporting to regulatory authorities if the SAE is 
considered a Suspected Adverse Reaction and is not listed in the current Investigator’s 
Brochure (ie, an unexpected event). 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
26 
 
To ensure consistency of AE and SAE causality assessments, investigators should apply 
the following general guideline: 
Fatal: 
Adverse event resulted in death. 
Unrelated: 
Another cause of the adverse event is more plausible; a 
temporal sequence cannot be established with the onset of the 
adverse event and administration of the investigational 
product; or, a causal relationship is considered biologically 
implausible. 
Possibly Related: 
There is a clinically plausible time sequence between onset of 
the adverse event and administration of the investigational 
product, but the adverse event could also be attributed to 
concurrent or underlying disease, or the use of other drugs or 
procedures. Possibly related should be used when the 
investigational product is one of several biologically plausible 
adverse event causes. 
Definitely Related: 
The adverse event is clearly related to use of the investigational 
product. 
Pregnancy 
Before study enrollment, subjects must agree to take appropriate measures to avoid 
pregnancy. However, should a pregnancy occur (including female partners of male 
subjects), the subject will consent to provide follow-up information regarding the outcome 
of the pregnancy and the health of the infant until 30 days old.  
Although pregnancy itself is not regarded as an AE, the outcome will need to be 
documented.  Report any pregnancy that occurs in a subject or subject’s partner from the 
time of consent to 30 days after the last dose of study drug.  Record any occurrence of 
pregnancy on appropriate case report form and fax it to Pharmacyclics Drug Safety, or 
designee, within 24 hours of learning of the event.  The pregnant female will be followed 
for outcome, which is defined as elective termination of the pregnancy, miscarriage, or 
delivery of the fetus.  For pregnancies with an outcome of live birth, the newborn infant 
will be followed until 30 days old by completing the Pregnancy Report Form Part II.  
Any congenital anomaly/birth defect noted in the infant must be reported as an SAE.  . 
 
A patient must immediately inform the investigator if the patient or patient’s partner 
becomes pregnant from the time of consent to 30 days after the last dose of study drug.  
Any female patients receiving PCI-32765 who become pregnant must immediately 
discontinue study drug. The investigator should counsel the patient, discussing any risks 
of continuing the pregnancy and any possible effects on the fetus.   

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
27 
 
Expedited Reporting Requirements for SAEs and Fatal or Life-Threatening 
Suspected Adverse Reaction Reports 
Investigators of studies conducted under an IND must comply with the following safety 
reporting requirements. 
The investigator must submit each IND safety report in a narrative format or on FDA 
Form 3500A or in an electronic format that FDA can process, review and archive.  A 
copy of this IND safety report must also be faxed to Pharmacyclics Drug Safety. 
The investigator must notify FDA and Pharmacyclics of any serious, unexpected, 
suspected adverse reaction observed during the conduct of the study as soon as possible 
but in no case later than 15 calendar days after becoming aware of the occurrence. 
The investigator must notify FDA and Pharmacyclics of any unexpected fatal or life-
threatening suspected adverse reaction as soon as possible, but no later than 7 calendar 
days after the Sponsor’s receipt of the information. 
Upon request from the FDA or Pharmacyclics additional data or information that the 
agency or Pharmacyclics deems necessary, must be reported as soon as possible but no 
later than 15 calendar days. 
 
 
 
FDA Contact 
 
 
 
 
Pharmacyclics Drug Safety Contact Information 
US Local Fax: 
1-760-268-6500 
US Toll Free Fax: 
1-877-676-0330 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
28 
 
13.0 Statistical Considerations  
The primary objectives of the statistical analysis will be (1) to demonstrate that there is a 
population of CLL cells that do not leave or take sanctuary outside of the peripheral 
blood system in solid lymphoid tissues, (2) to describe the longitudinal pattern in 
observed labeled cells and (3) to estimate rates of cell death. 
 
For purposes of statistical analysis, the experiment will be divided into two periods (with 
each period, itself, being divided into two subperiods):  Period 1 corresponds to the non-
interventional period when heavy water is consumed for 4 weeks (Period 1a) and then 
discontinued (Period 1b) so that a CLL cell disappearance rate can be tracked over time. 
 
Period 2 corresponds to the interventional period when Btk inhibitor (PCI-32765) is 
administered to subjects.  Assuming that the observed percentage of labeled cells (LC) 
reaches a peak at some point in time, T, then Period 2a is defined as the “pre-peak” sub-
period (i.e., all times t ≤ T) and Period 2b is the “post-peak” sub-period (t >T). 
 
Strictly speaking, Period 1 does not enter into any statistical analysis, as it only serves to 
provide labeling for the cells that allegedly remain and return and are retained in solid 
tissues.  However, Period 1 will permit definition of CLL cell birth rate and 
disappearance rate (death and permanent return to solid tissue from blood). 
 
An important part of the statistical analysis in Period 2 is characterization of the pattern 
of LC over time.  Patterns will be analyzed both visually and using more formal statistical 
methods in an exploratory manner.  First, the pattern will be smoothed using a cubic 
spline interpolation (SAS Graph, SAS Institute, Cary, NC).  This will facilitate the 
determination of shape and numbers and patterns of peaks by eliminating “noise” in the 
data.  Second, based on the patterns observed after smoothing, the data will be fit to 
candidate regression models (e.g., parabolas, mixtures of parabolas, piecewise curves, 
etc.) and the best fitting model will be identified using common statistical methods such 
as R2, Akaike’s information, etc.  All curves will be fit on individual subjects, thereby 
permitting subjects to vary in the pattern of their LC response curves. 
 
For example, one possible pattern would be an inverted-U parabola (Figure 1), where LC 
initially increases (presumably, as previously labeled CLL cells are simultaneously 
retrieved from solid tissue sanctuaries) and then declines (as the solid tissue sanctuaries 
have been depleted).  Another example would be a mixture of two inverted-U parabolas 
(Figure 2), whereby there are two distinct peaks, suggesting that retrieval of cells from 
solid tissue may take place at two distinct times.  Clearly, many other patterns are 
possible; however, the most likely candidate patterns are those with some form of initial 
upward slope, followed by a global peak, followed by a downward slope. 
 
Interim analyses and stopping rules: In order to monitor study progress and success, 
interim analyses will be done after the first 5 and the first 10 patients have been labeled 
and treated for 3 months.  The study will be placed on hold if the interim analyses 
determine that in less than 3 out of 5 patients or in less than 6 out of 10 patients, 
sufficient kinetic biomarker measurements can be obtained to accomplish the study 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
29 
 
objectives (for technical and other reasons, such as patient compliance). In this case, the 
study would need to be modified and can only be re-opened after a protocol revision that 
addresses such potential technical and/or logistical issues, which needs to be agreed upon 
among the Principal Investigators and the sponsor.   At that time, the investigators 
together with the sponsor will determine whether the data indicate that the objectives of 
the study can be met, or whether substantial changes to the protocol or data analyses 
would become necessary.  This interim analysis will, however, not place the patient 
accrual on hold, unless the interim analyses would come to the conclusion that the study 
needs to be modified. In addition, at any time during the trial, if we see more than 30% of 
patients experiencing grade 3-4 non-hematological toxicities, we will stop the trial. 
 
 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
30 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In general, no formal statistical inference is planned (i.e., hypothesis tests or confidence 
intervals).  The analysis is exploratory with the primary objective of discovering patterns 
of observed labeling that might provide further insights into the kinetics and biology of 
CLL. 
 
 
Figure 1 - An Inverted-U Parabola 
 
 
Figure 2 - Mixture of 2 Inverted-U Parabolas 
 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
31 
 
14.0 Sample Size Considerations 
This is primarily a (structured) exploratory study to discover patterns of cell kinetics in 
CLL.  There are no data from previous experiments that would permit specification of a 
formal alternative hypothesis for which a power or precision calculation could be made.  
The proposed sample size is 30 subjects of which it is expected that 18 (approximately 
60%) will be evaluable. An evaluable patient for the purpose of this protocol is primarily 
defined as a patient who has been able to adhere to all protocol-required procedures and 
blood draws during the heavy water labeling period, the resting period, and during 
treatment with PCI-32765. In addition, to be considered an evaluable patients, sufficient 
material with detectable levels of heavy water-labeled CLL cells must be recovered from 
the blood specimen by our colleagues at the Feinstein Institute and at KineMed, Inc. 
Specifically, it is expected that about 20% of subjects will exhibit a delayed release of 
labeled cells into the circulation in Period 1, which will manifest itself as an initially 
“flat” response; this number is based on available information from previous studies of 
this type.  These subjects will be excluded from both the Period 1 and 2 analyses, but 
may be analyzed separately.  In addition, it is anticipated that 20% of subjects will not be 
able to delay treatment for the desired 12-16 weeks post-labeling.  These subjects will 
also be excluded from both the Period 1 and 2 analyses, but their data may be analyzed 
and used separately.  The proposed sample size is consistent with other studies in CLL 
using similar methodologies.  If necessary, the overall accrual will be increased in order 
to yield at least 20 evaluable subjects. 
 
 
15.0 References 
 
1. 
Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (Campath-
1H) in patients who have failed fludarabine: results of a large international study. 
Blood. May 15 2002;99(10):3554-3561. 
2. 
Wierda W, O'Brien S, Faderl S, et al. A retrospective comparison of three 
sequential groups of patients with Recurrent/Refractory chronic lymphocytic 
leukemia treated with fludarabine-based regimens. Cancer. Jan 15 
2006;106(2):337-345. 
3. 
Ponader S, Chen SS, Buggy JJ, et al. The Bruton tyrosine kinase inhibitor PCI-
32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in 
vitro and in vivo. Blood. Feb 2 2012;119(5):1182-1189. 
4. 
O'Brien S, Burger JA, Blum KA, et al. The Bruton's Tyrosine Kinase (BTK) 
Inhibitor PCI-32765 Induces Durable Responses in Relapsed or Refractory (R/R) 
Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL): 
Follow-up of a Phase Ib/II Study. ASH Annual Meeting Abstracts. November 18, 
2011 2011;118(21):983-. 
5. 
Asher E, Payne CM, Bernstein C. Evaluation of cell death in EBV-transformed 
lymphocytes using agarose gel electrophoresis, light microscopy and electron 
microscopy. II. Induction of non-classic apoptosis ("para-apoptosis") by tritiated 
thymidine. Leuk Lymphoma. Sep 1995;19(1-2):107-119. 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
32 
 
6. 
Rocha B, Penit C, Baron C, Vasseur F, Dautigny N, Freitas AA. Accumulation of 
bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: 
minimal estimates of production and turnover rates of mature lymphocytes. Eur J 
Immunol. Aug 1990;20(8):1697-1708. 
7. 
Jones PJ, Leatherdale ST. Stable isotopes in clinical research: safety reaffirmed. 
Clin Sci (Lond). Apr 1991;80(4):277-280. 
8. 
Price JC, Guan S, Burlingame A, Prusiner SB, Ghaemmaghami S. Analysis of 
proteome dynamics in the mouse brain. Proc Natl Acad Sci U S A. Aug 10 
2010;107(32):14508-14513. 
9. 
Price JC, Holmes WE, Li KW, et al. Measurement of human plasma proteome 
dynamics with (2)H(2)O and liquid chromatography tandem mass spectrometry. 
Anal Biochem. Jan 1 2012;420(1):73-83. 
10. 
Price JC, Khambatta CF, Li KW, et al. The effect of long term calorie restriction 
on in vivo hepatic proteostatis: a novel combination of dynamic and quantitative 
proteomics. Mol Cell Proteomics. Dec 2012;11(12):1801-1814. 
11. 
Messmer BT, Messmer D, Allen SL, et al. In vivo measurements document the 
dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest. 
Mar 2005;115(3):755-764. 
12. 
Neese RA, Misell LM, Turner S, et al. Measurement in vivo of proliferation rates 
of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA. Proc 
Natl Acad Sci U S A. Nov 26 2002;99(24):15345-15350. 
13. 
Cheson BD, Bennett JM, Grever M, et al. National Cancer Institute-sponsored 
Working Group guidelines for chronic lymphocytic leukemia: revised guidelines 
for diagnosis and treatment. Blood. Jun 15 1996;87(12):4990-4997. 
14. 
Burger JA, Quiroga MP, Hartmann E, et al. High-level expression of the T-cell 
chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in 
nurselike cell cocultures and after BCR stimulation. Blood. Mar 26 
2009;113(13):3050-3058. 
15. 
Davis RE, Ngo VN, Lenz G, et al. Chronic active B-cell-receptor signalling in 
diffuse large B-cell lymphoma. Nature. Jan 7 2010;463(7277):88-92. 
16. 
Burger JA, O'Brien S, Fowler N, et al. The Bruton's Tyrosine Kinase Inhibitor, 
PCI-32765, Is Well Tolerated and Demonstrates Promising Clinical Activity In 
Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma 
(SLL): An Update on Ongoing Phase 1 Studies. Blood. November 19, 2010 
2010;116(21):32a. 
17. 
Honigberg LA, Smith AM, Sirisawad M, et al. The Bruton tyrosine kinase 
inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of 
autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A. Jul 20 
2010;107(29):13075-13080. 
18. 
Ponader S, Buggy J, O'Brien S, Wierda WG, Keating M, Burger JA. Bruton's 
Tyrosine Kinase Inhibitor PCI-32765 Abrogates BCR- and Nurselike Cell-
Derived Activation of CLL Cells In Vitro and In Vivo. Blood. November 19, 
2010 2010;116(21):26a. 
19. 
Advani RH, Buggy JJ, Sharman JP, et al. Bruton Tyrosine Kinase Inhibitor 
Ibrutinib (PCI-32765) Has Significant Activity in Patients With 
Relapsed/Refractory B-Cell Malignancies. J Clin Oncol. Oct 22 2012. 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
33 
 
20. 
Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and 
treatment of chronic lymphocytic leukemia: a report from the International 
Workshop on Chronic Lymphocytic Leukemia updating the National Cancer 
Institute-Working Group 1996 guidelines. Blood. Jun 15 2008;111(12):5446-
5456. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
August 15, 2014 
 
 
Protocol # 2012-0086 
 
 
34 
 
Schedule of Events 
 
 
Evaluation or 
Screening  
Treatment Plan 
  
  
Heavy Water (2H2O) 
Procedure 
(30 Days) 
Week 1 
Week 2,4 
Week 6 
Week 8 
Week 10 
Week 12 
Week 14 
Week 16 
Clinical 
Assessments 
Day -30 to 
-1 
Day 
1 
Day 14, 
Day 28 
Day 
42 
Day 
56 
Day 
70 
Day 
84 
Day 
98 
Day 
112 
Informed Consent 
 X 
  
  
  
  
  
  
  
  
Follow-up Visits 
with physical 
evaluationa, b,o 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOS/WHO Eval 
X 
  
  
  
  
  
  
  
  
Height/Weight 
X 
  
  
  
  
  
  
  
  
Concomitant 
Medications and 
Treatment 
X 
  
  
  
  
  
  
  
  
PT/PTT/INRc 
X 
  
  
  
  
  
  
  
  
Hematologyd and 
Serum Chemistrye 
X 
X 
X 
  
X 
  
X 
  
X 
Beta-2-
microglobulin and 
immunoglobulin 
levels 
X 
 
 
 
 
 
 
 
 
Blood Sample for 
heavy water testing 
  
X (on 
day 1, 
see r) 
Xq 
Xq 
Xq 
Xq 
Xq 
Xq 
Xq 
CLL Panelf 
X 
  
  
  
  
  
  
  
  
Bone Marrow 
Aspiration and 
Biopsyg 
X 
  
  
  
  
  
  
  
  
Electrocardiogramh 
X 
  
  
  
  
  
  
  
  
CT Scansi 
X 
  
  
  
  
  
  
  
  
Heavy Water 
(2H2O)  
  
Xj,k 
Xk 
  
  
  
  
  
  

Schedule of Events for 
Heavy Water and PCI-32765 
07-03-2013 
35 
 
Schedule of Events 
 
Treatment Plan PCI-32765 
 
Evaluation or 
 
 
Procedure 
 
 
 
Cycle 1 (weeks 1-4) 
Cycle 2 (weeks 6-8) 
Cycle 3  through 6 
(weeks 10-20) 
Cycle 6, 
9 
Cycle 12 
Cycles 
13 
through 36, and 
after cycle 36 
(see 
footnote 
“l”) 
 
         Week 1 
Week 2 
Week  4 
Week 6 
Week 8 
Week10 
Week12 
16, 20 
Week 
24, 36 
Week 48 
 
Clinical Assessments 
Day 1 
(+/-3 
days) 
Day 3  
(+/-1 Day) 
 
 
 
 
 
 
 
 
 
 
Follow-up Visits, with physical 
evaluationa, e, ,l 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
Serum Chemistrye and 
Hematologyd,  
X 
 
 
X 
X 
X 
X 
 
X 
 
X 
X 
PT/PTT/INRp 
 
 
 
 
 
 
 
 
X (only 
week 12) 
 
X 
X 
Blood Sample for heavy water 
testing 
X 
X 
 
Xq 
Xq 
Xq 
Xq 
Xq 
Xq (only 
week 12) 
 
 
 
CT Scanss 
 
 
 
 
 
 
 
 
X (only 
week 12) 
X 
X 
X 
PCI-32765 (3 x 140 mg =420 mg)m 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Allopurinol 300mgn 
X 
X 
 
X 
 
 
 
 
 
 
 
 
Immunoglobulin Levels and 
Lymphocyte Subpopulationf 
X 
 
 
 
 
 
 
 
X (only 
week 12) 
X 
X 
X 
Bone marrow aspirationt 
 
 
 
 
 
 
 
 
X (only 
week 12) 
X 
 
X 
Blood draw for research testingu 
X 
X 
 
 
X 
 
 
 
X (only 
week 12) 
 
 
 
Sputum Samplev 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Schedule of Events for 
Heavy Water and PCI-32765 
07-03-2013 
36 
 
a. 
Including vital signs. 
b. 
Patients will be seen at day 1 (+/- 3 days), day 14 (+/- 3 days), and day 28 (+/- 3 days) after beginning 2H2O ingestion for follow-up visits with clinical and laboratory 
assessment, which will include determining if there have been any problems with the ingestion of heavy water and assessment of other adverse events, assessment of 
concomitant medications, analysis, and discussion of intake logs and blood sampling (60ml) for 2H2O kinetic measurements. CBCs (white blood cell, hemoglobin, platelets and 
differential) will be monitored on days 1, 14, 28 (+/- 3 days), and then every 4 weeks (+/- 3 days) thereafter.  
c. 
Prothrombin Time/PT; Partial Thromboplastin Time/PTT, and International Normalized Ratio/INR. 
d. 
Hematology and serum chemistry: complete blood counts (white blood cell, hemoglobin, platelets and differential) will be monitored on days 1, 14, 28 (+/- 3 days) for the first 
four weeks and then every four weeks thereafter. 
e. 
Includes sodium, potassium, calcium, magnesium, phosphorus, BUN, creatinine, glucose, albumin, total protein, alkaline phosphatase, total bilirubin, ALT, and uric acid. 
f. 
IgG, IgM, IgA levels. Flow cytometry assessment for peripheral CLL cells and lymphocyte subpopulations (CD3, CD4, CD8, CD19, CD16/56 cells).  
g. 
Prior one month from registration.  Bone marrow will be evaluated by flow cytometry for clonality and for lGHV mutation studies (unless known), CD38 and ZAP-70 
expression, and cytogenetic and genomic abnormalities by FISH  
h. 
Electrocardiogram (prior one month from registration) will be required. 
i. 
CT of the chest, abdomen, and pelvis within 30 days before starting Heavy Water. 
j. 
Patient will be given 50ml 70% heavy water three times a day for the first 5 days (1-5). Patients will be given their first dose in clinic due to the possibility of lightheadedness 
or dizziness possible effect, and asked to avoid driving or performing potentially hazardous activities for one hour (labeling phase). Patients then will be given individual doses 
of heavy water to consume at home. 
k. 
Patients will be given individual maintenance doses of 60ml 2H2O daily (day 6-28) to be drunk at bedtime. At the end of the 4th week, patients will stop drinking 2H2O 
(washout phase) and be followed for 6-12 weeks until beginning treatment with PCI-32765. 
l. 
After beginning PCI-32765 therapy, patients will be seen for follow-up visits on day +1 (+/- 3 days), day 3 (+/-1 day), day +14 (+/-3 days), day +28 (+/-3 days) and every 2 
weeks thereafter for a period of at least equal in duration to that of the initial 2H2O washout phase of 6 to 12 weeks. These visits will include assessment of adverse events and 
concomitant medications.  After that, patients will continue to return to clinic each month (+/- 3 days) or will be seen by a local physician for follow-up visits with clinical and 
laboratory assessment, including vital signs and physical exam, and laboratory workup, including hematology and clinical chemistry evaluations until the end of 12 cycles.  If 
subjects stay on the study past 12 cycles, they will return for clinic visits for clinical assessment, including vital signs and physical exam, and laboratory workup, including 
hematology and clinical chemistry evaluations every 3 months (+/- 1 month) for cycles 13 through 36, and every 6 months (+/- 1 month) thereafter.  For patients that 
discontinue therapy with PCI-32765, an end of study visit at MD Anderson will be required. Patients will be followed for at least 2 months (+/- 1 month) after the last dose of 
PCI-32765 for assessment of adverse events. This will be done in a Leukemia Clinic visit that will be scheduled to occur at 1 month (+/- 10 days) after the last dose of PCI-
32765. 
m. Administer 3 140mg capsules orally once daily with 8 ounces of water; this will be continued daily. Treatment duration will be 12 cycles, with each cycle consisting of 28 days 
(336 days total). Treatment beyond cycle 12 is permitted if a patient continuous to demonstrate therapeutic benefit. 
n. 
Allopurinol at the dose of 300mg daily will be given during the first one or two weeks of treatment as standard tumor lysis prophylaxis. 
o. 
Patients are required to be seen at UT MD Anderson Cancer Center for screening period only. The follow-up visits with physical evaluation during weeks 1 through 16 can be 
done locally at the patient’s local doctor’s office. 
p. 
PT,PTT,INR, and bleeding time will be monitored after cycles 3 and 12 (+/- 1 week) and if patients stay on the study past 12 cycles, once every 12 cycles (+/- 1 month). 
q. 
Blood draws (60ml) at week 2 (+/- 3 days) and every 2 weeks (+/- 3 days) thereafter for a period of at least equal in duration to that of the initial 2H2O washout phase (6 to 12 
weeks) for 2H2O kinetic measurements. Data obtained from these blood samples will permit a determination of the slope and rate of the appearance of previously labeled CLL 
cells in the blood, and the slope and rate of the disappearance of labeled cells from the blood. 
r. 
Anti-coagulated blood sample (50ml) and coagulated blood sample (10ml) for baseline 2H2O studies on the day of study initiation. 
s. 
CT scans of the chest, abdomen, and pelvis after cycles 3 or 6, and 12. Beyond cycle 12, CT scans are at physicians discretion. 
t. 
Bone marrow will be evaluated by flow cytometry for clonality and for amount of CLL cell infiltration, including detection of minimal residual disease after 3, 6, and 12 cycles 
(+/- 1 month) of therapy with PCI-32765. Bone marrow testing at later time points is at the treating physician’s discretion. 
u. 
Blood draws using one 2.5ml PaxGen tube will be collected in cycle 1, week 1, days 1 (+/- 3 days) and 3 (+/- 1 day), week 4 (+/- 3 days), and week 12 (+/- 3 days), for research 
testing. 
v. 
One Sputum sample will be collected on day 1 (+/- 3 days) of beginning therapy with PCI-32765 for research testing.