A Phase I-II Study of Busulfan-fludarabine Conditioning and T-cell Depleted Allogeneic Stem Cell Transplantation for Patients With Advanced Hematologic Malignancies
Phase: PHASE1
Conditions: Leukemia, Lymphoma, Myeloma
Sponsor: University of Chicago

 
1
IRB# 12-0132 
THE UNIVERSITY OF CHICAGO 
 
DIVISION OF MEDICINE 
 
A phase I-II study of busulfan-fludarabine conditioning and T-
cell depl eted all ogeneic st em ce ll transplantation for patients 
with advanced hematologic malignancies 
 
 1.0 
OBJECTIVES 
 
 2.0 
BACKGROUND 
 
 3.0  
BACKGROUND DRUG INFORMATION 
 
4.0 
PATIENT ELIGIBILITY 
 
 5.0  
DONOR SELECTION, STEM CELL SOURCE AND TREATMENT PLAN 
 
 6.0  
PRETREATMENT EVALUATION 
 
 7.0  
EVALUATION DURING STUDY 
 
 8.0 
CRITERIA FOR STUDY EVALUATION  
 
 9.0  
STATISTICAL CONSIDERATIONS 
 
10.0 
DATA AND PROTOCOL MANAGEMENT 
 
11.0 
CRITERIA FOR REMOVAL FROM PROTOCOL 
 
 12.0 REPORTING REQUIREMENTS 
 
 13.0 REFERENCES 
 
 
 APPENDIX A: 
TREATMENT SCHEME 
 
 
 APPENDIX B: 
GVHD CRITERIA 
 
 
 Version 08/11/06, 09/13/2011, 02/20/2013 

 
2
Principal Investigator  
 
 
 ___________________________ 
 Andrew Artz MD 
 
 
 CO- INVESTIGATORS 
 
 ___________________________ 
 
 
__________________________ 
 Wendy Stock, M.D.  
 
 
 
 
Samir Undevia, M.D. 
 
 ___________________________ 
 
 
__________________________ 
 Richard Larson, M.D.  
 
 
 
 
Toyosi Odenike, M.D. 
 
 ___________________________ 
 
 
__________________________ 
 Chris Daugherty, M.D. 
 
 
 
 
Sonali Smith, M.D. 
 
 ___________________________ 
 
 
__________________________ 
 Amittha Wickrema, M.D.  
 
 
 
Susana Marino, M.D., Ph.D.  
 
 __________________________ 
 
 
__________________________ 
 Todd Zimmerman, M.D. 
 
 
 
 
Lucy Godley, M.D., Ph.D. 
 
 __________________________ 
 
 
__________________________ 
 Hongtao Liu, M.D., Ph.D  
 
 
 
Justin Kline, M.D. 
 
__________________________  
 
 
 Michael Bishop, M.D. 
 
 
 
 
 
 
 
University of Illinois at Chicago 
 
Principal Investigator:  
 
_______________________ 
 David J. Peace, MD 
 
 

 
3
PROTOCOL ABSTRACT 
Protocol: 
A phase I-II study of busulfan-fludarabine conditioning and T-cell depleted allogeneic stem cell 
transplantation for patients with advanced hematologic malignancies 
 
Principal Investgator: 
 
Andrew Artz, M.D. 
 
Patient Eligibility: 
Diagnosis:  
Phase I portion: 
 Relapsed or refractory acute myelogenous or lymphoid leukemia.   
 Chronic myelogenous leukemia in accelerated phase or blast-crisis. 
 Recurrent or refractory malignant lymphoma or Hodgkin’s disease 
 Recurrent or refractory multiple myeloma. 
 Chronic lymphocytic leukemia, relapsed or with poor prognostic features. 
 Myeloproliferative disorder (polycythemia vera, myelofibrosis) with transformation 
 Myelodysplastic syndromes with more than 5% blasts. 
Phase II portion: 
 AML with active disease or beyond CR2 
 MDS with more than 5% blasts 
Zubrod performance status   2 (See Appendix B). 
Life expectancy is not severely limited by concomitant illness. 
Adequate cardiac and pulmonary function. Patients with decreased LVEF or PFTS will be 
evaluated by cardiology or pulmonary prior to enrollment on this protocol. 
Calculated creatinine clearance >50 ml/min. 
Serum bilirubin   2.0 mg/dl, SGPT <3 x upper limit of normal 
No evidence of chronic active hepatitis or cirrhosis.HIV-negative Patient is not pregnant Patient 
or guardian able to sign informed consent. 
Matched sibling donor, 1 Antigen mismatched relative or matched unrelated donor. 
 
Treatment Plan: 
See appendix A 
PATIENT EVALUATION: 
See section 6 and section 7 
 
Miscellaneous Information: 
 
 

 
4
 Objectives: 
1. To establish the maximally tolerated dose (MTD) of intravenous busulfan (Busulfan®) in 
combination with fludarabine  as conditioning regimen for transplantation with in-vivo T-cell 
depletion 
2. To evaluate disease free and overall survival after this conditioning regimen in patients with 
advanced AML and MDS. 
3. To evaluate potential pharmacogenomic determinants of toxicity of this regimen. 
4. To evaluate potential pharmacogenomic determinants of efficacy of this regimen. 
 
 
Statistics: 
The phase I portion of the trial will use the modified continual reassessment method (CRM) to  
determine the maximum tolerated dose (MTD).  The primary endpoint for the phase II trial will be the
disease-free survival rate (DFS) at one year, and a Simon two-stage design will be used to test  
the null hypothesis that the one-year DFS rate is 25% against the alternative that it is at least 40%.   
Adverse events will be monitored, and multiple regression, logistic regression, and Cox regression 
analyses will be performed to examine the association between GSTA1 enzymatic activity and 
busulfan metabolic rate, GSTA1 enzymatic activity and the incidence of toxicity, and pharmaco- 
genomic predictors of DFS, respectively. 
 
 

 
5
1 
OBJECTIVES 
4.1 
To establish the maximally tolerated dose (MTD) of intravenous Busulfan 
(busulfex®) in combination with fludarabine  as conditioning regim en for 
transplantation with in-vivo T-cell depletion 
4.2 
To evaluate disease free and overal
l survival after 
this conditioning 
regimen in patients with advanced AML and MDS. 
4.3 
To evaluate potential pharm acogenomic de terminants of toxicity of  this  
regimen. 
 
4.4 
To evaluate potential pharmacogenomic determinants of efficacy of this 
regimen. 
 
 
5.0 BACKGROUND 
5.1 
Allogeneic transplantation in advanced
 hematologic 
malignancies. 
 
Allogeneic transplantation has been extensively used for the treatment of advanced 
hematologic malignancies and results in durable remissions in approximately 30% 
of patients. Besides patient related factor s, the outcome of transplantation depends  
on (1) the condition ing regimen, (2) type of donor (HLA-matched vs -mismatched 
or unrelated), (3) Type of GVHD prophylaxis.  
 
5.2 
Conditioning regimens for a
llogeneic transplantation. 
Historical and institutional results. 
 
Traditionally, total body irradiation or high doses of busulfan in combination with 
high doses of cyclophospham ide have been th e m ainstay for conditio ning in 
allogeneic transplan tation. But more recen
tly reduced intensity co
nditioning 
regimens have been utilized in an attem
pt to reduce early regim
en related 
toxicity.1-3 Such regimens assure reliable 
engraftment in recipients of HLA-
identical transplants, and prolonged diseas e free survival has been demonstrated 
in a proportion of patients with end-stage malignancies.  
We have  evaluated a com
bination of fl udarabine and m elphalan as originally 
pioneered by Giralt et al. 1 Using this regim en, we obtained approximately 30% 
disease-free surviva l, 30% tr eatment re lated m ortality and observ ed a high  
incidence of  extensive chronic GVHD.  Di
sease free survival  was obtained in 
approximately 20-30% of the patients.4 In a subsequent study, at the University of 
Chicago (IRB 11300 A), we have continued
 to use the sam
e condition ing 

 
6
regimen, but em ployed Ca mpath 1H (alem tuzumab), a T-cell depleting agent, 
instead of m ethotrexate for GVHD prophylaxis.
5 This c onditioning results in  
excellent engraftment, much decreased toxicity, near abs ence of chronic GVHD 
and excellent initial survival. Unfortunately, recurrence rates are high in patients 
with advanced leukemia and lymphoma and long-term disease free survival in this 
patient group is likely to be in the range of 20% to 35%.5-7 (and manuscript submitted) 
 
Our experience indicates that treatment related mortality not only depends on the 
conditioning regimen, but also depends on the toxicity of the medications used for 
GVHD prophylaxis an d on the efficacy
 of GVHD prevention. In
deed, the 
fludarabine melphalan conditioning regimen has considerably more toxicity when 
tacrolimus and methotrexate are used for GVHD prophylaxis, than with the use of 
Campath and a short course of tacr
olimus. Vice versa, tacrolimus and 
methotrexate are less ef ficient in preven ting GVHD than alem tuzumab. So m ore 
patients develop acute a nd chronic GVHD with tacrolim
us-methotrexate. This  
also results in an increase in treatment related mortality. 
 
5.3 
Busulfan for conditioning in transplant. 
To address the problem of delayed disease recurrence  after fludarabine-melphalan 
conditining and GVHD prophylaxis with al
emtuzumab, we are interested in 
studying th e efficacy and treatm
ent relate d mortality associated with a m
ore 
intensive conditioning regimen, but similar alemtuzumab based GHVD prophylaxis 
regimen. 
 
Busulfan (1,4-bis-(m ethanesolfonoxyl) butane)  is a bifunctional alkylating agent, 
which was first described by Haddow and Timm is.8  Since the demonstration of its 
potent antitumor effects, it has been used extensively for the treatment of malignant 
disease, especially hematologic malignancies and myeloproliferative syndromes. Its 
use was for a long tim e limited to low dose or al therapy with pa lliative intent, and 
frequent monitoring of the blood counts was routinely recommended. The advent 
of some 2 t o 3% of patients developing busulfan-induced pulmonary fibrosis, as 
well as occasionally severe, som etimes ev en irreversible myelosuppression after  
prolonged adm inistration effectively de terred dose escalation beyond 8-10 m
g 
daily. 
 
In 1974, however, Santos and Tutschka invest igated the use of busulfan to create a 
murine m odel of aplas tic anem ia.9  Subsequently, the expe rience gain ed in th is 
model system was used to introduce hi gh-dose combination chemotherapy based 
on oral busulfan for pr
etransplant conditioning of non-hum
an prim ates and 
thereafter patients undergoing both 
autologous and allogeneic m
arrow 
transplantation.10-17 Since then, high-dose busulfan, most commonly in combination 
with cyclophospham ide (Bu/CY), has pr
oven to be an effective an
tileukemic 
regimen when used in conjunction with 
autologous or allogeneic hem atopoietic 
stem cell support and is comparable in efficacy to TBI containing regimens.18 
 

 
7
High-dose busulfan thera
py has several advantag
es for use in
 m arrow 
ablation/pretransplant treatm ent.  Firs
t, when using chemotherapy alone for 
conditioning of patients undergoing m
arrow transplantation, 
one avoids the 
dependence on a radiation unit with, us
ually, lim ited capacity to  delive r th e 
necessary treatment on a fixed schedule.  Second, a radiation-based regim en can 
only be delivered to patient
s who have not been previo
usly irradiated.  Many 
patients with lym phoma, Hodgkin’s dis ease and leukem ia have had extensive 
radiation previously for cont rol of locally agg ressive disease in sites s uch as the 
mediastinum, neck, and CNS.  Additio
nally r adiation as p
art of  the  
pretransplantation conditioni ng regim en may cause irreve
rsible and often fatal 
toxicity in s uch cases.  However, a m
ajority of previously radiated patients can 
safely receive a busulfan-based regim en, provided that the previous acute radiation 
toxicity (usually within the f irst 2-4 months after r adiation therapy) has subsided.  
Third, in selected p atients who suf fer recurrent leukemia after allogeneic m arrow 
grafting, a second marrow transplant may still offer a chance for long-term  disease 
control or even cure.
19 Due to su
bclinical (irrever sible) toxicity, a TBI-based 
regimen can only be utilized once
 in a patien t’s lif etime, whereas com bination 
chemotherapy can be e
mployed following a previous TBI-based regim
en.  
Busulfan-based chemotherapy may, therefore, serve as a valid alternative. 
 
5.3.1 Rationale for a Parenteral Formulation of Busulfan in Marrow 
Transplantation 
 
Oral busulf an has, unfortunately, several serious shortcom ings.  When used in 
high-dose combination regimens, serious si de effects in the liver and lungs are 
often encountered 10;20;21 Several investigators have reported veno-occlusive 
disease (VOD) of the liver leading to fata l liver failure, as the m ost serious side 
effect.11;13;14;17.  Neurologic disturbances such as grand m al seizures, and severe 
nausea and  vom iting are also freq
uently en countered.22-25 It is im possible to  
predict which patients  will d evelop liv er failure, and it is furth
er unknown 
whether the liver failure is due to tox icity from the systemic busulfan or w hether 
it is mainly due to a first-pass phenomenon.  Based on the limited information that 
is available regarding busulfan pharmacokinetics, it appears however that patients 
who absorb a large fraction of the inge sted dose, with a prolonged high busulfan 
plasma concentr ation, will b e a t incre ased risk f or dev eloping ser ious sid e 
effects.14;17;26;27 Another disadvantage with oral 
busulfan is that patients who 
develop severe nausea and vomiting shortly (within one-half to two (1/2-2) hours) 
after a dose has b een delivered will lo se part or all of the dos e, and it will be 
virtually impossible to accurately d etermine how much of t he dose has been lo st 
in a vomiting subject.  Furthermore, the intestinal resorption of any delivered drug 
may be influenced by the patient’s 
nutritional status, and by concurrent 
administration of other drugs affecting the intestinal microenvironment, as well as 
by whether the patien
t has eaten
 in cl
ose proxim ity to ingestion of the 
administered drug dose and, finally, by th
e inherent biological variability in 
intestinal absorption between different patients. Due to these uncertain ties, oral 
administration of high-dose busulfan carries  with it an inherent safety problem 

 
8
both from  the potential danger of inadvert
ent overdosing  with a risk for leth al 
toxicities, as well as from the hazard of  suboptimal underdosing the patient with 
an inadve rtently high  p otential f or recur rent o r persisten t m alignancy af ter the  
marrow transplant.   
 
Busulfan dissolved in DMA/PEG400/dext rose (Busulfex®)  has been recen
tly 
approved for conditio ning in transplantation  and has replaced o
ral busulfan  
because of  its reliable dosing
 and 
its m ore predictable 
intrapatient 
pharmacokinetics.  A multi-ins titutional trial of IV Busulfan in combination with 
cyclophosphamide as pretransplant c
onditioning therapy in patients with 
advanced hem atologic m alignancies indicated  that the  saf ety prof ile is grea tly 
improved when the busulfan is adm inistered IV as com pared with the standard  
oral busulfan for mulation.  In particular, the incidence of serious, lifethreatening 
or lethal veno-occlusive disease of the liver was  reduced to <20% of wha t would 
be expected  f rom using oral Busulf an in high- dose pre transplant con ditioning 
therapy in p atients with adva nced hematologic malignancies.28 Additional safety 
information was obtained a series of CML patients undergoing HSCT after this 
regimen when the busulfan is given as a standardized dose based on PK 
parameters. These lim ited data sugges t an “optim al” dose-exposure (“area under 
the curve” AUC) for Busulfex, below a
nd above which there is increased 
treatment failure.29  
 
5.3.2 Rationale for using fludarabine in combination w
ith IV 
busulfan 
 
The acute toxicities of high-dose or
al busulfan in com
bination with 
cyclophosphamide are well known.  Chief a mong the described adverse events is 
VOD which is dose-related and only rarely seen in patients whose plasma AUC is 
less than 1,500 µMol-m in.17 Rece ntly, Mcdonald et al dem
onstrated that the 
hepatic toxicity of the comm only used cyclophosphamide-TBI regimen is mainly 
due to cyclophosphamide metabolites and suggested that toxicity may be reduced 
by replacing cyclophospham ide w ith less toxic alternatives.
30 It is likely that 
cyclophosphamide m etabolites also contri bute to the toxicity of the busulfan-
cyclophosphamide regimen, a situation in which cyclophospham ide metabolism 
and toxicity is influenced by the prior administration of busulfan.31 Fludarabine is 
an effective drug again st hematologic malignancies and appears less toxic than 
cyclophosphamide in many studies.1   
As discussed above, the com bination of fludarabine and intravenous m elphalan 
has been actively explored by our section as a less intens ive preparative regimen 
that allows engraftm ent of allogen eic progenitor cells from
 both related and 
unrelated d onors and acceptable toxicities in
 older and m
edically debilitated  
patients.11 We are now interested in establishing the MTD of intravenous busulfan 
combined with fludarabine in com bination with T-cell deplet ed transplantation. 
Others have already evaluated this com bination in different doses, with different 
GVHD prophylaxis and with other form
ulations. Slavin et al, studied the 
combination of low dose busulfan and 
fludarabine in patients who were not 

 
9
otherwise elig ible f or myeloablative conditioning therapy and reported 
encouraging results.32  The Seattle group combined fludarabine with oral busulfan 
in myeloablative doses and demonstrated an acceptable toxicity profile.33 Finally, 
Russell et al. have used IV Busulf an with fludarabine as conditioning therapy for 
patients with hem atologic disorders under going allogeneic HSCT.  They used 
once daily adm inistration of both Fl udarabine (50 m g/m2) and IV Busulfan (3.2 
mg/kg BW) for four da ys in com bination with cyclosporine-m ethotrexate based 
GVHD prophylaxis. The obtained safety info rmation indicated, that this regim en 
is well tolerated without unexpected side effects, and allowing for c
onsistent 
engraftment and good antitum
or effect.
34 Further, they obtained lim
ited 
pharmacokinetic (PK) infor
mation, which support the previous notion that 
busulfan in this dosing interval displays  linear pharmacokinetics. A similar study 
was reported by the MD Anderson group.35 
 
5.3.3 Rationale for further dose escalation studies of busulfan.  
Current conditioning regimens have lim ited cure rates in patien ts with advanc e 
hematologic m alignancies. There are 
considerable data suggesting a dose-
response curve for cure rates with 
busulfan conditioning in hem
atologic 
malignancies.29;36 Furth er dose-es calation m ight therefore be associated with 
higher cure rates. 
In the past dose escalation to an AU C above 1500 m cgr.min/L.dose (when four  
divided doses were given) was associat
ed with an unacceptab
le increase in  
VOD.14;17;36-39 But there are at several observations  that suggest that further dose 
escalation of the Busulfan com ponent m ight be possible w ith the fludarabine-
busulfex-alemtuzumab regimen. 
 
1. Intravenous busulfan is less hepatoto
xic than oral busulfan. 2. Fludarabine-
busulfan combination is likely to be considerably less hepatotoxic than the 
commonly used cyclophospham
ide-busulfan com
bination, w
here 
cyclophosphamide metabolites contribute to regimen related toxicity. 
3. Alemtuzumab based GVHD prophylaxis is likely to be less hepatotoxic than 
the commonly used cyclosporine methotrexate based GVHD prophylaxis. 
 
5.3.4 Rationale for test dose and AUC based dosing. 
 
The use of a test-dose followed by freque nt pharmacokinetic sampling will allow 
the assessment of individual  patient pharmacokinetics and calculation of the dose 
necessary to achieve an  intended AUC with subsequent doses. 27;40 IV Busulfex 
has  linear pharmacokinetics27;29;34;41 and does not accumulate even after repeated 
daily dosing. On the other hand, there con
tinues to be consider able interpatient 
variability in pharm acokinetics and the AUC c annot reliably be predicted from  
the administered doseOn the other hand, there is considerable evidence to indicate 
that busulfan AUC is c losely associated  with toxicity and, AUC should guide 
dose escalation studies rather than administered dose.   
In this study , all patien ts will receiv e an initial test dose of  0.5 m g/kg IV over 3 
hours. Pharmacokinetic sampling will be pe rformed at four tim e intervals after 

 
10
completion of  the inf usion and use d to calcula tes trea tment doses ne cessary to  
achieve an intended AUC. The treatm ent doses will be administered as four daily 
doses. Pharmacokinetic sampling will be performed with the first and fourth dose 
to conf irm the r elationship be tween pr edicted and obtained AUC. The target 
starting da ily AUC (4800 um ol-min) is s lightly lower tha nt the m edian AUC 
(4897 umol-min) observed in the MD A nderson study with a fixed dose of 130 
mg/kg.35 One should however also take in
to account th e additional one-tim e 
busulfan exposure (approxim ately 600 um ol-min) from  the test dose in our 
patients. Therefore, even at the starting target AUC, cum
ulative busulfan 
exposure for our patients  at 19800 umol-m in, should be slightly higher than the 
median exposure in the MD Anderson study of approximately 19600 umol-min. 
 Blood samples will be stored on each
 of the patients for subsequen
t 
pharmacogenomic studies. In patients w ith active AML and MDS tum or samples 
will be stored as well for subsequent pharmacogenomic studies.  
 
5.4 Donor 
type 
 
The outcome of allogeneic transplantation is determ ined to a large degree by the 
choice of stem  cell donor. The best outco
mes are obtained with the use of 
syngeneic donors (i.e. identical twins). 
Grafts from  such donors are accepted 
without risk for graft failure or graft versus host disease. 
Among siblings, the probability of being id entical for all m ajor histocompatibility 
(HLA) antigens is approxi
mately 25%. Allogeneic 
matched (HLA-identical) 
siblings can be identified for approxim ately 20-30% of the population.  Seventy 
percent of patients who m
ight otherwise benefit from  alloge neic hem atopoietic 
stem cell transplantation lack a suitably  matched related donor.  Alternative donor 
sources include partially m
atched rela ted donors, unrelated
 volunteers, and 
previously banked placental blood. Recipients of unrel ated donor transplantation 
will be included in this  study as they have been in all our previous s
tudies. As in 
our previous studies, we will use m
olecular typing (DNA sequencing ) of 10 
relevant HLA alleles (A, B, C, DR, DQ) for determining donor/recipient matching.   
 
5.5 
Rationale for the current study 
In a previous study, we 
utilized fludarabine-m elphalan-campath, followed by 
transplantation of allogeneic m atched sibling or m atched unrelated donor stem 
cells. Engra ftment was excellent  a nd treatm ent rela ted morta lity m uch reduced  
compared with prev ious experience.42 One year survival was im proved compared 
with historical controls and m ost encour agingly, the quality of life of transplant 
recipients was excellent because of the near complete absence of chronic GVHD.   
Unfortunately the recu
rrence ra te in pa
tients with  ad vanced he matologic 
malignancies was high.  In the current study w
e continue to use cam path GVHD 
prophylaxis because of its excellent tolerance and efficacy. We propose to combine 
it with a
 f ludarabine busulf an condition ing regim en and individua
lized 
pharmacokinetics based dosing. We hypothe
size that intensif
ication of the 

 
11
conditioning regimen will result in impr oved disease control. We hypothesize that 
AUC based dosing will lim
it toxicity and 
that furth er d ose es calation of this  
regimen will be possible.  The phase I portion of this study will enroll patients with 
a variety of hematologic malignancies. However, we anticipate that, as in previous 
studies, approximately 50% of our patients will have AML and MDS. The phase II 
portion of this study will be lim ited to pa tients with advanced AML and MDS, a 
patient population where we have previous ly observed a poor outcom e.  Patients 
with other disease stages or histologies will then be entered on other protocols. 
5.6 
Pharmacogenomic determinants of hepatic toxicity 
The major metabolic pathway of busulfan is through sulfonation by transfer of a sulfur 
group from Glutathione through Gl utathione S-transferases (GST ), a process that occurs 
mainly in the liver. T
hereafter, the liphoph ilic interm ediary undergoes oxidation by 
cytochrome P450 enzymes before excretion. 31 Two fam ilies of GSTs are distinguis hed, 
soluble and microsomal GST’s. The soluble GST’s in humans comprise at least 16 genes 
grouped in 8 classes and are the GS
T’s involved in busulfan m
etabolism.43 The 
microsomal GST’s are s tructurally unrelated to the soluble GST’s and of no relevance to 
busulfan metabolism. 
 At least eight subtypes of soluble GST have been described, each with specific affinities 
and more or less tissue restricted expressi on. The pivotal GST for busulfan m etabolism is 
GST A1.44  GSTM1 accounts for approxim ately 48% of busulfan metabolism and a third 
GST, GSTP1 accounts for approximately 18%.
45;46 GST A1 is exp ressed mainly in th e 
liver, but can also be detected in the blood. 
In-vitro experim ents indicate considerable 
inter-individual variation in hepa tic expression of GST A1 which correlates closely w ith 
busulfan metabolizing potential.44;47 Surprisingly GSTA1 leve l in the bloodstream as
 
measured with a commercial kit als o correlates with busulfan metabolizing activity and 
may represent a surrogate of GSTA1 activity in the liver.  
SNP analysis dem onstrates the ex istence of at least six polym orphic sites in  the GSTA1 
promoter, and the existence of five different alleles. 44;48  Based on the relations between 
the haplotypes, two subclasses of GSTA1 can  be distinguished, GSTA1A and GSTAA1B 
with respectively two and three variants. According to Coles, allelic variation in GSTA1 
genotype accounts to a large extent for the hi ghly variable activity of GSTA1. But the in-
vivo relation between GSTA1 genotypes and busulfan pharm
acokinetics, remains to be 
studied.   
In vitro data suggest that  sulfonated bus
ulfan m etabolites as well as GSH depletion 
contribute  to toxicity of hepatocytes.49 Others have shown that overexpression of GSTA1 
in cell lines, results in protec tion against busulfan induced cell cycle arrest and ind uction 
of tissue factor expression, thus suggesting a potential link between  GST A1 expression 
and busulfan induced toxicity.
50 It is therefore conceivab le that differences  in  GSTA1  
activity caused by GSTA1 polym orphisms  affect busulfan toxi city independent of  their 
postulated effects of busulfan pharmacokinetics. 
GST-M1 is another hepatic GST that is re
sponsible for a substantial proportion of 
busulfan metabolism. The GST M1 gene is known to be highly polymorphic with deletion 
of either or both genes at varying but signifi cant frequencies in different ethnic groups. In 
a recent study of chi
ldren with  thalassem ia receiv ing busulfan cyclophosph
amide 
conditioning, GSTM1 null genotype was the mo
st i mportant risk factor for  VOD.
51 

 
12
Busulfan clearance was higher in p atients with null genotype and first dose steady state 
concentration was lower. The m
echanism by which GSTM1 null genotype causes 
increased risk for VOD is probably com
plex a nd m ay be related to direct toxicity of 
busulfan metabolites (perhaps due to r
eciprocally in creased GS TA1 activity),
52 
intrahepatic GSH depl etion or interac tion of GSH depl
etion and cyclophospham ide 
toxicity. Co nfirmation of  these res ults af ter c onditioning with busulf an-fludarabine, is  
likely to provide important new information on this issue. 
 
5.7 
Pharmacogenomic determinants of  drug efficacy: 
In addition to stud ies relating GST polym orphism to toxicity, we will, in patients with 
leukemia, study leukemia blasts to determine the relation between polymorphisms relating 
to busulfan and/or fludarabine metabolism and efficacy. For this purpose, we will study a 
sample of leukemia cells obtained from the bone marrow. Such samples will be obtained 
from AML patients participating in the phase I and or phase II portion of the protocol.  
 
Although resistance to fludarabine may occur by several means, one proposed 
mechanism of cellular and clinical resistance is reduced intracellular drug 
accumulation53.  The early steps of plasma membrane transport, nucleoside 
phosphorylation, and nucleotide dephosphorylation determine intracellular 
accumulation of fludarabine and its metabolites. Fludarabine is administered as the 
5'-nucleotide monophosphate and is converted to the nucleoside by the activity of 
serum phosphatase and ecto-5'nucleotidase (CD73). As fludarabine is a hydrophilic 
compound and does not readily cross plasma membranes by diffusion, the presence 
of functional nucleoside transporters that accept fludarabine as a permeant is 
required for cellular entry at rates sufficient to achieve cytotoxic levels of 
intracellular metabolites.  Three human (h) nucleoside transporter proteins, 
equilibrative nucleoside transporter 1 (hENT1), equilibrative nucleoside transporter 
2 (hENT2), and concentrative nucleoside transporter 3 (hCNT3), have been shown 
to mediate cellular entry of fludarabine54-56.  
Within the cell, fludarabine requires anabolism to the active 5'-triphosphate 
(fludarabine triphosphate) to exert its cytotoxic effects. The rate-limiting step in this 
process is the conversion by deoxycytidine kinase (dCK) of free nucleoside to the 
5'-nucleotide monophosphate (Plunkett 91). Directly opposing dCK activity are the 
intracellular 5'-nucleotidases, including the high-Km 5'-nucleotidase (CN-II) and 
deoxynucleotidase-1 (dNT-1) (Galmarini 01).   
In order to identify molecular markers of potential predictive value in AML 
patients, we will evaluate AML patients who had not previously been treated with 
fludarabine to determine 1) the cellular expression of genes encoding nucleoside 
transporter proteins and enzymes mediating fludarabine metabolism, and 2) the role 
of polymorphisms in these genes. 
 
RNA will be extracted from leukemia cells in bone marrow specimens by 
quantitative real-time PCR.  We will then analyze the relationships between RNA 

 
13
expression, germ line and somatic polymorphisms in the gene described above, and 
time to disease progression following fludarabine therapy.  
 
Similarly, due to the role of GSTs in the inactivation of busulfan, we will evaluate 
1) the expression of GST genes in leukemia cells, 2) its correlation with germ line 
and somatic GST polymorphisms, and 3) the correlation among GST expression, 
polymorphisms and time to disease progression. 
 
 
6.0 
BACKGROUND DRUG INFORMATION 
6.1 Tacrolimus 
(Prograf) 
    
TACROLIMUS is a macrolide compound with potent immunosuppressant 
properties.                           
    
DOSING INFORMATION:  TACROLIMUS is usually given intravenously 
initially in doses of 0.03 mg/kg/day for 3 days, followed by conversion to oral 
therapy (0.09 mg/kg twice daily); dose adjustments are required in patients with 
hepatic dysfunction.                    
HUMAN PHARMACOLOGY:  The oral absorption of TACROLIMUS iserratic 
and incomplete; absolute bioavailability is approximately 25%; peak serum levels 
are seen 1 to 4 h after an oral dose, and therapeutic serum concentrations have 
ranged from 0.2 to 6 ng/mL;TACROLIMUS is extensively metabolized in the 
liver, with only small amounts of unchanged drug (2% or less) being recovered in 
the urine; the elimination half-life of TACROLIMUS is approximately 10 h.    
CAUTIONS:  Common adverse effects of TACROLIMUS areheadache, 
hyperesthesia, tremors, circumoral numbness, insomnia, nausea, abdominal 
discomfort, and appetite       
insomnia, nausea, abdominal discomfort, and appetite changes; all of these effects 
occur primarily with IV TACROLIMUS and are more frequent during combined 
use of TACROLIMUS and CYCLOSPORINE; other adverse effects include 
nephrotoxicity, hyperkalemia, hyperuricemia, hyperglycemia, dysphasia, 
photophobia, flushing, and lymphoproliferative disorder; unlike 
CYCLOSPORINE, hirsutism, gingival hyperplasia, and hypertension are 
generally not seen with TACROLIMUS; combined therapy with 
CYCLOSPORINE has resulted in increases in cyclosporine serum levels and 
more severe nephrotoxicity.           
6.2 Busulfan
 
 
BUSULFAN is an alkylating agent that interferes with DNA replication and 
transcription of RNA and ultimately results in the disruption of nucleic acid 
function. 
DOSING INFORMATION:  In transplantation, doses of 1 mg/kg PO are repeated 
to a usual cumulative dose of 16 mg/kg. The usual dose for IV Busulfan is 80% of 
the equivalent PO dose. 

 
14
HUMAN PHARMACOLOGY: Busulfan is rapidly absorbed from the 
gastrointestinal tract, and measurable blood levels are obtained within 0.5-2 hours 
after oral administration.  Within 3 minutes after IV administration in rats, 90% of 
the drug disappears from the blood; similar rapid decreases in blood concentrations 
have been reported in man.  Busulfan is reported to be extensively metabolized; 12 
metabolites have been isolated, but most have not been identified.  The drug is 
slowly excreted in the urine, chiefly as methanesulfonic acid.  Ten to 50% of a dose 
is excreted as metabolites within 24 hours. 
CAUTIONS : Dose limiting toxicity is hematological.  Long term therapy has been 
associated with pulmonary fibrosis and an Addison's like syndrome. Seizures have 
been reported after high dose Busulfan used for transplantation. Dilantin is often 
administered preventively.  
6.3 Fludarabine 
 
Fludarabine is the 2-fluoro, 5-phosphate derivative of vidarabine.     
DOSING INFORMATION:  Doses of 25 mg/m(2)/day (30-minute infusion) for 5 
days every 4 weeks has been effective previously treated patients with chronic 
lymphocytic leukemia; in non-Hodgkin's lymphoma, a loading dose of 20 mg/m(2) 
intravenously followed by a continuous intravenous infusion of 30 mg/m(2)/24 
hours for 48 hours, has been effective; dose reductions are suggested in renal 
insufficiency. 
PHARMACOKINETICS:  Following intravenous administration, fludarabine 
phosphate is rapidly dephosphorylated to 2-fluoro-vidarabine, which subsequently 
enters tumor cells and is phosphorylated to the active triphosphate derivative; peak 
plasma levels of 2-fluoro-vidarabine have ranged from 0.3 to 0.9 mcg/mL 
following a short infusion of 25 mg/m(2) fludarabine; 24% of a dose of fludarabine 
is recovered in the urine as 2-fluoro-vidarabine; the elimination half-life of 
2-fluoro-vidarabine is 9 hours. 
CAUTIONS:  Myelosuppression, particularly neutropenia, is the predominant 
adverse effect; a severe neurotoxicity has been observed, mainly with higher doses; 
other adverse effects include nausea, vomiting, diarrhea, stomatitis, skin rash, and 
somnolence; pneumonitis has been reported in 1 patient.        
CLINICAL APPLICATIONS:  Intravenous fludarabine has been highly effective 
in heavily pretreated patients with chronic lymphocytic leukemia; the drug has also 
produced responses in patients with non-Hodgkin's lymphoma and acute leukemia; 
however, neurotoxicity has been a major concern, even with low doses, and more 
studies are needed to clarify its ultimate place in therapy.           
6.4 
CAMPATH- 1H (Alemtuzumab) 
CAMPATH-1H is a humanized monoclonal antibody directed against CD52, an 
epitope that is abundantly expressed on T- and B- lymphocytes, but not on NK 
cells. It has been extensively used for the prevention of GVHD both in vitro and 
in vivo. 
DOSING INFORMATION:  In transplantation, daily doses of 20 mg are repeated 
for up to five times. 

 
15
HUMAN PHARMACOLOGY:  Campath is extensively bound to circulating 
CD52 in the serum as well as present in unbound form. The half life of Campath 
is prolonged and free Campath can be detected for several weeks after 
administration. Campath is usually administered intravenously, but recent data 
indicate that subcutaneous administration is associated with a decreased incidence 
of severe reactions. In some studies Campath has been admixed with the stem cell 
infusate. 
CAUTIONS: The infusion of Campath H1 has been associated with fever,nausea, 
headache, vomiting, rash, chills and rigor. Occasionally hypotension and 
bronchospasm have been reported. This can be managed by adequate 
premedication. 
 
7.0 P
ATIENT ELIGIBILITY 
7.1 
Patients with the following diseases:  
Diagnosis:  
Phase I portion: 
 Relapsed or refractory acute myelogenous or lymphoid leukemia.   
 Chronic myelogenous leukemia in accelerated phase or blast-crisis. 
 Recurrent or refractory malignant lymphoma or Hodgkin’s disease 
 Recurrent or refractory multiple myeloma. 
 Chronic lymphocytic leukemia, relapsed or with poor prognostic features. 
 Myeloproliferative disorder (polycythemia vera, myelofibrosis) with 
transformation 
 Myelodysplastic syndromes with more than 5% blasts. 
Phase II portion: 
 AML with active disease or beyond CR2 
 MDS with more than 5% blasts 

 
16
7.2 
Zubrod performance status   < 2  
7.3 
Life expectancy is not severely limited by concomitant illness. 
7.4 
Adequate cardiac and pulmonary function. Patients with 
decreased LVEF or PFTS will be evaluated by cardiology or 
pulmonary prior to enrollment on this protocol. 
7.5 
Calculated creatinine clearance >50 ml/min . 
7.6 
Serum bilirubin  < 2.0 mg/dl, SGPT <3 x upper limit of normal 
7.7 
No evidence of chronic active hepatitis or cirrhosis. 
7.8 HIV-negative 
7.9 
Patient is not pregnant 
7.10 Patient or guardian able to sign informed consent. 
 
8.0 DONOR 
SELECTION, STEM CELL SOURCE AND 
TREATMENT PLAN. 
8.1 DONOR 
SELECTION 
When possible, an HLA compatible sibling will be used as a donor. For patients 
who do not have an HLA-compatible sibling, an unrelated donor will be 
identified. 
 
In case of unrelated donor transplantation, high resolution DNA-based HLA 
typing will always be performed for HLA A, B, C and DR antigens   
8.2 
HEMATOPOIETIC STEM CELL SOURCE  
Donor stem cells will be the preferred source of HSC in all cases. Based on 
donor’s preference, bone marrow stem cells may be utilized in some recipients.  
 
9.0 TREA
TMENT PLAN 
 
9.1.1 All patients shall be registered with the Data Management 
Office. 
 
Complete all sections of the Registration Form located on the University of 
Chicago Cancer Research Center web site at http://www-uccrc.uchicago.edu/ and 
send the completed form by facsimile to 773-702-8855. 
9.1.2 Conditioning regimen  
 

 
17
Day  
Clonazepam Fludarabine 
Busulfan 
Bu 
pharmacokinetics 
Campath 
Test Dose 
- 
PO 
 
 
 
 
- 
PO 
 
0.5 mg/kg 
x 6 
 
- 
PO 
 
 
 
 
Treatment Doses 
-7 PO 
25 mg/m2  
 
20 mg 
-6 
PO  
25 mg/m2 
AUC based 
x 5 
20 mg 
-5 
PO  
25 mg/m2 AUC 
based 
 
20 mg 
-4 
PO  
25 mg/m2 
AUC based 
(x 5*) 
20 mg 
-3 
PO  
25 mg/m2 
AUC based 
x 5* 
20 mg 
-2 
PO 
   
 
-1 
 
   
 
0 
 
   
 
 
Comments: 
Busulfan administration has been associated with a low incidence of seizures and 
dilantin is routinely used for seizure prophylaxis. Dilantin use is discouraged in this 
protocol because exposure to dilantin can affect Busulfan metabolism.57 
We recommend benzodiazepines e.g clonazepam (klonopin ®) 1 mg PO TID 
Scheduling: 
Busulfan test dose: 
The test dose can be scheduled any day within 8 days prior to admission. Clonazepam 
will be started with an evening dose, the day prior to the test dose and two doses one day 
after test dose. Busulfan test-dose will be based on actual body weight and will be 
accompanied by extensive pharmacokinetic studies. This dose will be given as an 
outpatient. 
Patients will be admitted in the evening of day –8, to start fludarabine on day -7  
 
Fludarabine: 
Fludarabine dosing will be based on actual body weight. Fludarabine will be infused over 
30 minutes before busulfan treatment dose. 
 
Busulfan treatment doses: 
 
On each day of treatment, busulfan will be administered through a fresh infusion line at a 
steady rate over 3 hours. Infusion will start in the morning around 5 AM and will be 
completed around 8 AM, allowing ample time for pharmacokinetic sampling in the 
afternoon.  
*Pharmacokinetic studies will be performed on the day 1 dose and again on the day 4 
dose. For logistical reasons (week-end, holidays), day 3 pharmacokinetics may be 
performed instead of day 4. Busulfan doses may be further adjusted based on levels 
obtained with treatment dose 1. 
 
 

 
18
Campath 
All patients will receive premedication for Campath as per current standards at the UC 
transplant unit. 
 
9.1.3 Dose schedule (see also statistical plan)  
The targeted daily AUC of busulfan will be determing using the modified continual 
reassessment method.  
 
Target AUC’s are the following: 
Dose level 0: 4800 mcgr.min/L.dose  
Dose level 1: 5800 mcgr.min/L.dose 
Dose level 2: 6800 mcgr.min/L.dose 
Dose level 3: 7800 mcgr.min/L.dose 
Dose level 4: 8800 mcgr.min/L.dose 
Dose level 5: 9800 mcgr.min/L.dose 
 
We do not anticipate further dose-escalation beyond dose level 5. If such dose-escalation 
were to be considered, we would submit an amended protocol to the IRB. 
 
 
9.1.4 Bone marrow/stem cel infusion 
On day 0 the stem cell product will be infused according to BMT unit policy. 
Bone marrow, PBSC will not be further processed except in the case of ABO-
incompatibility. In such instances, red blood cells will be removed from bone marrow 
products or from PBSC products containing excessive amounts of RBC (as per transplant 
policy). 
All stem cell products procured through the NMDP will be done so in strict compliance 
with the protocols, policies, and procedures established by the NMDP.  
 
 
9.1.5 GVHD prophylaxis  
 Tacrolimus 0.03 mg/kg/day IV CI over 24 hr from 4 PM day -2 until engraftment or 
when patient is able to take PO, then tacrolimus 0.09 mg/kg PO in 2 divided doses.  
Tacrolimus should be given at full dose to maintain levels of 5-15 ng/mL through day 
100. Thereafter tacrolimus will be tapered by 20% every week unless the patient has 
developed GVHD. In recipients of mismatched or unrelated donor transplants, tacrolimus 
will be continued until day 180. Thereafter tacrolimus will be tapered by 20% every week 
unless the patient has developed GVHD. 
 
9.1.6 Supportive care:  
Infection prophylaxis and supportive care will be as BMT unit policy.  No routine growt 
factor support will be administered. Growth factor support will be considered in case of 
delayed engraftment (ANC < 0.5x109/L on day 12) 
 

 
19
9.1.7 Back-up bone marrow harvest (only for recipients of 
mismatched or unrelated donor transplants) 
For patients transplanted in remission, a backup bone marrow or peripheral blood stem 
cell collection will be considered within two weeks prior to hospital admission as long as 
the overall marrow cellularity is greater than 30%. All backup harvests will be 
cryopreserved after processing for buffy coat without any further manipulation. The 
backup stem cells will be given if deemed clinically necessary in the event of failure to 
engraft by day +35, graft rejection after day +35, or graft failure after day +35. 
 
 
10.0 PHARMACOKINETIC STUDIES 
Busulfan will be infused with a fresh infusion set whenever pharmacokinetic 
assessments will be performed. The tubing will be primed with the busulfan, and the 
entire volumeplus a volume of normal saline flush equal to the priming volume will 
be infused at a constant rate into a central venous catheter over the 3-hour infusion 
time. Sampling will be done with the test dose, and with the first and fourth 
treatment doses. If, for logistical reasons, sampling on day 4 is not possible, then day 
3 may be used as the time point for the second sample. Samples will be taken from a 
peripheral vein (through the same line after additional flushing) into prechilled 
heparin tubes with plasma separation within 30 minutes. 
 The actual start and stop times of busulfan will be recorded, as will the exact times 
when the blood levels are drawn. Plasma will be separated by centrifugation at 2500 
rpm for 10 minutes at 4°C within 1 hour, placed in cryogenic vials, and analyzed or 
stored at -40°C until analyzed by a validated high-pressure chromatography in the 
Pharmacokinetics core lab at the University of Chicago Cancer Center. 
10.1 Timing of samples: 
10.1.1 Test dose: 
Blood samples (5 mL) will be taken before Bu infusion, at 175 minutes (five 
minutes before completion), 240 minutes (1 hour after completion) 300 minutes (  
2 hours after completion), 360 minutes (3 hours after completion) and  420 
minutes (4 hours after completion) of the test dose  
10.1.2 Treatment doses 1 and 3 (or 4): 
Blood samples (5 mL) will be taken before Bu infusion, at 175 minutes (five 
minutes before completion), 240 minutes (1 hour after completion) 300 minutes (  
2 hours after completion), 360 minutes (3 hours after completion of the treatment 
dose 1 (day -6) and treatment dose 4 (day -3) (or in selected patients, treatment 
dose 3, day –2) 
 
The Software program WinNonLin Pro ® will be used for calculating AUC. 

 
20
11.0 PRETREA TMENT EVALUATION 
11.1 RECOMMENDED EVALUATION OF THE PATIENT 
The pre-transplant evaluation of the patient will follow recommendations as per 
transplant policies (on website). The following list can be used for guidance.   
 
 Complete history and physical examination 
 Bone marrow biopsy and aspirate with leukemia markers,  
 cytogeneticsCBC, platelets, differential, reticulocyte count, PT and PTT 
 Chest X-ray and PFTs with DLCO 
 Baseline EKG,  
 MUGA with measurement of LVEF 
 Chemistry profile with complete liver function panel. 
 Complete urinalysis 
 HLA Class I and class II (molecular typing in case of unrelated donor 
transplantation) 
 ABO and Rh typing 
 Serum titers for CMV, HSV,HIV antibody, hepatitis screen and any other 
assays required for donor work-up by transplant unit policy (usually based on 
FDA recommendations and/or recommendations from federation for 
accredition of cell therapy, FACT) 
 Peripheral blood for chimerism studies. 
 Lumbar puncture for cell count, protein, glucose, and cytology in patients with 
ALL and high grade NHL, or if prior history of CNS involvement.  
 CT scan of the chest (with IV contrast) for all patients. 
 CT of abdomen and pelvis for lymphoma/Hodgkin’s disease  patients only. 
 Quantitative immunoglobulin, and bone survey for multiple myeloma patients 
only. 
 Urine pregnancy test if female 
 Any additional tests that may be required for clinical care and described in the 
BMT unit policies. 
 20 cc of blood and 5 cc of bone marrow will be stored on all recipients to be 
used for research purposes. (including pharmacogenomic assays) 

 
21
11.2  RECOMMENDED EVALUATION OF THE DONOR 
The pre-transplant evaluation of the donor will follow recommendations as 
per transplant policies (on website). The following list can be used for 
guidance.  
 Complete History and Physical Examination 
 CBC, platelets, differential, reticulocyte count, PT and PTT 
 Chest X-ray 
 EKG 
 Electrolytes, BUN, Creatinine, SMAC, magnesium 
 Complete Urinalysis 
 HLA Class I and  Class II typing (molecular typing in case of unrelated donor 
transplantation) 
 ABO and Rhesus typing 
 Serum titers for CMV, HSV, EBV 
 HIV serology, Hepatitis screen and any other assays required for donor work-up 
by transplant unit policy (usually based on FDA recommendations and/or 
recommendations from federation for accredition of cell therapy, FACT) 
 Peripheral blood for chimerism studies. 
 20 cc of blood will be stored on all donors to be used for research purposes. 
 20 cc of blood will also be stored on each day of stem cell collection 
 
12.0 EVALUATION DURING STUDY 
 
12.1.1 EVALUATION DURING THE FIRST 100 DAYS 
 
Evaluation during the first 100 days will be done as per routine for the allogeneic 
transplant patient (see transplant policies). 
. 
Restaging of disease and engraftment studies will be performed between day 25 
and day 35. 
12.1.2 DAY 100 EVALUATION (to be completed day 84-100) 
The day 100 evaluation of the patient will follow recommendations as per 
transplant policies (on website). The following list can be used for guidance. 
 
12.1.2.1 Review of Systems and Physical examination 
12.1.2.2 CBC, differential, platelets, electrolytes, BUN, 
Creatinine, Magnesium, glucose, SMAC,  
12.1.2.3 Bone marrow biopsy and aspirate with cytogenetics  
12.1.2.4 Peripheral blood chimerism studies. 
12.1.2.5 Bone marrow aspirate and biopsy 
12.1.2.6 restaging of disease as indicated by disease specific 
testing. 
 
 

 
22
12.1.3 EVALUATION DAYS 100-365 (patient without chronic 
GVHD) 
The one year evaluation of the patient will follow recommendations as per 
transplant policies (on website). The following list can be used for guidance. 
 
12.1.3.1 PE and screening labs at least monthly through day 365 
12.1.3.2 Restaging of disease approximately 3 months, 6 months, 
9 months and one year after BMT. 
12.1.3.3 Follow-up for patients with chronic GVHD as per the 
chronic GVHD protocol. 
 
 
12.1.4 ANNUAL EVALUATION 
The yearly evaluation of the donor will follow recommendations as per transplant 
policies (on website) . The following list can be used for guidance. 
 
12.1.4.1 Review of systems and physical examination 
12.1.4.2 Schirmer's test  
12.1.4.3 Chest X-ray 
12.1.4.4 Pulmonary function tests 
12.1.4.5 CBC, differential, platelets, electrolytes, BUN, 
Creatinine, glucose, SMAC, magnesium. 
12.1.4.6 Bone marrow biopsy and aspirate with cytogenetics and 
chimerism studies. 
12.1.4.7 Thyroid function tests 
12.1.4.8 Restaging of leukemia by bone marrow aspirate and 
biopsy with cytogenetics and chimerism studies. 
12.1.4.9 restaging of disease. 
 
12.1.5 blood and bone marrow samples 
Samples of blood and/or bone marrow will be obtained at the following time points: 
 Prior to admission  (BM and blood) 
 On day 0 (Blood) 
 On day 7 (Blood) 
 On day 14 (Blood) 
 On day 28 (BM and blood) 
 Day 50 (Blood) 
 Day 75 (Blood) 
 Day 100 (Blood) 
 Day 150  (BM and blood) 
 At relapse (BM and blood) 
 One year and yearly thereafter (BM and blood). 
 
These samples will be stored for our ongoing studies of MRD and for assessment of 
immune reconstitution as well as chimerism. Chimerism will be determined by molecular 
analysis of peripheral blood samples. 

 
23
 
An  initial blood sample will also be stored for pharmacogenomic studies. 
 
As of February 2013, after IRB approval,
 cryopreserved pre-tr ansplant and post-
transplant recipient specim ens and pre-transplant donor sp ecimens collected under this 
protocol will be used to evaluate the e
ffects of m inor histocom patibility antigen s and 
major histocompatibility antigens (HLA) mismatches on tran splant outcomes. Genomic, 
proteomic, functional tests, an d other tests will be performed 
to iden tify predic tors of  
compatibility between patient and donors. Th
ese predictors m ay he lp future studies 
exploring id entification of  the m ost suitab le do nor(s) for p atients planning allogeneic 
hematopoietic cell tran splantation. This w ill be done for pr otocol IRB 11300A and IRB 
12-0132. 
   
 
 
DNA isolation will be performed from 1 ml of EDTA-blood sample from each patient 
sample using the Magnapure Compact system for DNA isolation (Roche 
Applied Science). 
 
Sample shipment information 
3-5 ml of patient blood sample should be drawn in a purple top EDTA tube.  Samples 
should NOT be spun down.  Samples should be shipped in appropriate packaging by 
overnight delivery at room temperature.  If a sample cannot be sent the day it is drawn, it 
can be stored refrigerated at 4°C for 2-3 days prior to shipment.  Samples may be shipped 
to the laboratory on Monday through Thursday.  Samples should not be shipped on 
Friday as the laboratory cannot receive samples on the weekend.  All samples need to be 
appropriately labeled with patient name/ID and protocol number.  In addition, all samples 
should be accompanied by paperwork that specifies the sample ID, protocol number, 
physicians name and contact information (address, phone number and fax number) to 
whom the result needs to be reported to.  Samples should be shipped to the University of 
Chicago Genetic Services Laboratory at the following address:  
 
University of Chicago Genetic Services Laboratory 
Department of Human Genetics 
The University of Chicago 
5841 S. Maryland avenue, Room L038 
M/C 0077 
Chicago, IL 60637 
 
phone: 773-834-0555 
fax: 773-834-5337 
 
Protection of confidentiality 
Patient samples will be processed and genotyped at the University of Chicago Genetic 
Services Laboratory which is a CAP and CLIA certified laboratory that routinely 
performs genetic testing of multiple disorders on a clinical basis.  The laboratory 

 
24
therefore has all necessary procedures related to protecting of patient confidentiality.  All 
samples will be processed by trained technologists and results reported only to the 
patient's physician or other appropriate health care person.  All patient samples will have 
a protected identification code that will be used. 
 

 
25
Table I: Schedule of tests (The following table can be used for guidance. Some individualization may be necessary) 
 
Tests & Observations 
Prior to 
Study 
During 
conditioning 
Day of 
Transplant 
Day 28 (± 1 wk) 
Post Transplant 
Day 100,   (± 1 
wk) 
Day 180 (± 1 wk) 
Post-Tx 
Follow-up** 
History and Progress Notes 
X 
 
X 
X 
X X 
X 
Physical Examination 
X 
X 
X 
X 
X X 
X 
Pulse, Blood Pressure 
X 
 
 
  
X 
X 
Height/Weight X 
X  
  
X 
X 
Performance Status 
X 
 
X 
X 
X X 
X 
 
 
 
 
 
 
 
 
Toxicity Assessment 
 
 
X 
X 
 
 
 
AGVHD Assessment 
 
 
X 
X 
X X 
 
CGVHD Assessment 
 
 
 
 
 X 
X 
Lumbar puncture  
E 
 
 
 
 
 
 
Surveillance Tests 
EKG 
X 
 
 
 
 
 
 
PFTs 
X 
  
 
 
 
X 
Thyroid function tests 
 
  
 
 
 
X 
Schirmer test  
 
  
 
 
 
X 
MUGA (or 2-D Echo) 
X 
 
 
 
 
 
 
Staging 
Chest x-ray, PA & Lateral 
X 
 
 
 
  
X 
CT/MRI scan chest/abd/pelvis 
X 
  
 
D 
D 
D 
Bone Marrow Asp & Bx 
X 
 
 
X 
X X 
X 
Laboratory Studies 
CBC, Differential, Platelets 
X 
X 
X 
X 
X X 
X 
Serum Creatinine, BUN 
X 
 
 
X 
X X 
X 
Serum Electrolytes 
X 
 
 
X 
X X 
X 
AST, ALT, Bilirubin 
X 
 
 
X 
X X 
X 
LDH 
X 
  
X 
X 
X 
X 
Urinalysis 
X 
 
 
 
 
 
 
serum or u-HCG (for pre-menopausal 
females) 
X 
 
 
 
 
 
 
Hepatitis Screen, CMV Ab, HIV, EBV, 
HSV-I 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serologies & HLA Typing  
X 
 
 
 
 
 
 
Pharmacokinetics 
 
X 
 
 
 
 
 
Chimerism 
 
Peripheral blood (20 cc min.)** 
X 
 
X 
X 
X X 
X 
Donor peripheral blood (5 cc) 
X 
 
 
 
 
 
 
Immune reconstitution 

 
26
Tests & Observations 
Prior to 
Study 
During 
conditioning 
Day of 
Transplant 
Day 28 (± 1 wk) 
Post Transplant 
Day 100,   (± 1 
wk) 
Day 180 (± 1 wk) 
Post-Tx 
Follow-up** 
Peripheral blood (20 cc min.) 
X 
 
X 
X 
X X 
X 
Optional Tests (i.e. dependent on patient approval) 
MRD  (WT1 or IgG gene) 
 
 
 
 
 
 
 
Peripheral blood (20 cc min.) 
X 
 
X 
X*** 
X X 
X 
Bone marrow aspirate (8 cc) 
X 
 
 
X 
X X 
X 
Pharmcogenomics  
X 
 
 
 
 
 
 
  Blood (20 cc) 
X 
 
 
 
 
 
 
  Bone Marrow (5 cc) 
X 
 
 
 
 
 
 
 
** At  one-year post-transplant, and then yearly thereafter for a maximum of 5 years from study entry, and at relapse. 
*** Additional samples on  day 7, 14,  
D For lymphoma patients. Other tests may be indicated in myeloma (e.g MRI or bone scan) 
E: Only for pts with ALL, High grade lymphoma or history of CNS involvement. 
 

 
27
13.0 CRITERIA FOR STUDY EVALUATION 
 
Relapse will be recorded by the day of initial detection of malignant cells, if these cells 
were on subsequent testing confirmed to be increasing in number. The molecular 
detection of MRD will not be taken into account for the definition of clinical recurrence. 
The diagnosis of disease recurrence will be based on clinical and pathological criteria. 
 
Toxicity will be scored according to NCI/CTC version 3 
(http://ctep.cancer.gov/reporting/ctc.html). Any grade 5 (fatal) toxicity will be considered 
a DLT. In addition grade 4 toxicities will be considered DLT with the exception of the 
following: Grade 4 hematologic toxicities will not be DLT. Grade 4 infections will not be 
DLT. Any grade 4 toxicity that can be attributed to infection will not be considered DLT. 
 
To assess severity of GVHD we will use the Seattle criteria (Bearman et al, Blood 85, 
2005, 1995). In this system, mild VOD is clinically obvious, but does not require 
treatment and resolves completely. Moderate VOD requires treatment such as diuretics or 
pain medications, but resolves completely. Severe VOD requires treatment and does not 
resolve completely. Severe VOD will be considered DLT. 
 
 Acute GVHD will be scored according to the criteria proposed by Przepiorka et al.58 
Chronic GVHD will be scored according to appendix C. Limited Chronic GVHD is 
defined as GVHD with limited skin involvement only or presenting with liver function 
abnormalities only. All other presentations of chronic GVHD are defined as extensive 
and will require treatment. 
 
High risk extensive chronic GVHD is characterized by the presence of  thrombocytopenia 
(<100,000/mm3).59 
 
 
 
The diagnosis of Veno-occlusive disease will be based on Baltimore criteria as follows: 
development of hyperbilirubinemia with serum bilirubin >2mg/dl with any two of the 
following symptoms: ascites, painful hepatomegaly and unexplained weight gain >5% 
from baseline within 20 days of BMT. Whenever possible, a presumed diagnosis of VOD 
will be confirmed by liver biopsy.60 
 
Engraftment will be defined as per IBMTR and NMDP guidelines. Cytogenetic and 
chimerism studies will be performed to confirm donor origin. 
  
Failure to engraft will be defined as lack of evidence of hematopoietic recovery (ANC < 
500/mm3 and platelet count < 20,000/mm3 ) by day +35, confirmed by a biopsy revealing 
a marrow cellularity < 5%. Graft failure will be defined as initial myeloid engraftment by 
day +35, documented to be of donor origin, followed by a drop in the ANC to < 500/mm3 
for more than three days, independent of any myelosuppressive drugs, severe GVHD, 
CMV, or other infection.  
 

 
28
Graft rejection will be defined as graft failure with documentation of return of recipient 
hematopoiesis as determined by cytogenetic and/or chimerism studies. 
 
 
14.0 ST ATISTICAL CONSIDERATIONS 
 
 
Phase I 
 
For the phase I portion  of the study, we will em
ploy the modified continual reas sessment method  
(CRM) to determ ine the m aximum tolerated dose (Faries, 1994; Goodm an et al., 1995)
61;62. Dose-
limiting tox icity (DLT) will b e as  defined und er 13.0 and, occurring within the f irst 21 days  after 
transplant.  Dose cohorts of size th
ree w ill be u tilized and we will targ et the 25th percen tile o f the 
tolerance distribution, i.e., the dose level producing DLT in 25% of  the patient population.  Briefly, the  
modified-CRM design utilizes a one-parameter, logistic regression model for the dose-toxicity curve 
 
                                                           
)
3
exp(
1
)
3
exp(
)
(
x
x
x
p






,                 (1) 
 
where
)
(x
p
 is the probability of DLT at dose level .x  A prior distribution is assumed for the parameter
and the first three patients are assigned to the lowest dose level.  Af ter outcomes are observed for  
these patients, the posterior distribution for  is calculated and the estim ated toxicity probabilities are 
updated.  T he next cohort of patients  are assigned to the dose level cl osest to the targeted percentile, 
with the restriction that doses can
not be esca lated m ore that one le vel at a tim e.  Enrollm ent is 
continued until a p re-determined total num ber of patients have been stu died, whereupon the MTD is  
derived based on the final, estimated dose-toxicity curve.   
 
In our trial, the f irst cohort of three patients will be enrolled at dose level 0 and after all three of these 
patients hav e been f ollowed to day 60 the observed nu
mber of  DLTs will be  used to up date the  
posterior distribution and determine the dose level fo r the succeeding  cohort.  Sin ce we anticip ate an 
accrual rate of approximately th ree patients per month, it is likely that  all three patients from a given  
cohort will not have been followed to 60 days before 
the next eligible patient becom es available.  In 
this c ase w e will assig n such patients ( termed excess p atients) to th e curr ent d ose leve l; o nce the  
original three patients p ass the 60 -day landmark, we will up date the prediction probabilities using all 
available data.  However, so as not to bias the procedure, if excess patients have not been followed for a 
full 60 days, we will not include 
their toxicity outcom es in the updated analysis , even if a D LT has 
been observed.  A total of 30 patients will be enrolled in the phase I trial.   
 
Because delayed VOD ( day 30 and day 60) of moderate  severity has been obser ved in two of the 12 
initial patients on study, the observation perio
d for su ccessive patients will be extended to 6
0 days 
before dose escalation. This will lead to accru
al of a higher num ber of patients per dose level and 
therefore we anticipate up to 50 patients in the phase I trial. 
 
Addendum June 2006: Pharm acokinetics for the initia l 20 patients was perform ed in the cancer center 
core facility at UC. For logistica l reasons and for more intensive monitoring another laboratory will be 

 
29
used and technical m odifications in sampling have been introduced. Therefore accrual will res ume at 
dose level 0. This will result in accrual of an additional 20 patients to the protocol. 
 
 
 
Phase II 
 
The primary endpoint for the phase II portion of the trial is the disease- free survival rate (DFS) at one 
year defined as the proportion of patients alive and without evidence of recurrence one year after the 
initiation of therapy.  All patients will be followed for a minimum of one year and we will test the null 
hypothesis that the DFS rate at twelve months is < 25% versus the alternative that it is at least 40%.  A 
Simon (1989), optimal two-stage design will be em ployed in which 29 patien ts are enrolled in th e first 
stage.63 (Patients treated at the MT D during the phase I portion of the study will be included in the 
phase II trial.)  If the number alive and without evidence of recurrence at  twelve months is 7 or fewer, 
the study will be terminated for lack of efficacy.  Otherwise, subject to the monitoring described below 
for graft failure, liver toxicity, and GVHD, an additional 43 patients will be enrolled for a total of 72.  If 
22 or fewer patients are disease free at 12 months the regimen will be rejected, whereas if 23 or more of 
the to tal 72  patients (>  32%) are disease free, the regim
en w ill be considered worthy of further 
evaluation in phase III trials.  This design has an 
- level of 10% and a power of 90% under the 
alternative hypothesis that the true 12-m onth DFS rate is 40%.   The probability of early term ination 
when the 12-month DFS rate is 25% is 0.56.  However,  to avoid a long suspensi on of the trial, accrual 
will continue into the second stage while the on e-year outcomes in the 29 first stag e patients are being 
accumulated.   
 
 
In addition, the incidence of graf t failure, liver toxicity, and high- risk, extensive chronic GVHD at 18 
months will be closely monitore d and evaluated.  For the prim ary endpoint of DFS, we will employ a  
Simon (1989) two-stage design. Graft failure, liver t oxicity, and GVHD will be m onitored at specified 
intervals as  descr ibed below.  An independe
nt Data an d Saf ety M onitoring C ommittee will be  
established to review the data and make recommendations regarding the continuation or discontinuation 
of the tr ial. This comm ittee is organized by  the University of Chicago Cancer Center (High risk pr
otocol 
monitoring committee) Since the assu mptions regarding efficacy and toxicity are the  same, the related  
donor and unrelated donor subgroups will be combined for all analyses. 
 
  
   Adverse Event Monitoring 
 
Graft failure and liver toxicity will be monitored and early termination of the trial will be considered if 
there is evidence that the graft failure rate is >1 0% or the ra te of liver toxicity is >15%.  Specifically, 
for graft failure the da ta will be reviewed after successive cohorts of 10 patients have been treated and 
we will consider terminating the trial if 4 of the first 10, 6  of the first 20, 7 of 30, 9 of 40, 10 of 50, 12  
of 60, or 13 of 70 have graft failure.  For liver toxicity the trial will be stopped if 5 of the first 10, 7 of 
the first 20, 9 of 30, 11 of 40, 13 of 
50, 15 of 60, or 17 of 70, develop liv er toxicity.  W ith regard to 
high-risk, extensive GVHD, we will review
 the data after the first 10, 20, and 30patients with 
successful engraftment and without evidence of  relapse reach their 18-month landm ark.  (By t he time 
30 patients have reached this point, all 72 patients will have almost surely been accrued.)  Evidence that 
the rate of high-risk, extensive 
GVHD is >40% will lead to cons
ideration for early stopping.
  
Specifically, if 8 of the first 10, 13 of the first 20, or 17 of 30 such patients develop G VHD at any time 

 
30
prior to 18 months we will cons
ider term inating the trial.  Each  of these th ree s topping guid elines 
approximates a Pocock (1977) 64 group sequential m onitoring boundary for a one-sided, overall alpha  
level of 0.10.   
 
    Additional Statistical Analyses 
 
At the conclusion of the phase II tr ial, 90% confidence intervals will be generated for the DFS rate at 
12 m onths, as well as for the incidence of graft 
failure, and the incidence of active and extensive 
GVHD.  Kaplan-Meier (1958) estimates of disease-free and overall surv ival rates will be calculated, 65 
and the m edian disease-free and ov erall survival times and their as sociated 90% confidence intervals 
derived using the m ethod given in Brookmeyer and Crowley (1982). 66 Descriptive statistics r elated to 
the frequency of adverse events, including liver toxici ty, and of changes in laboratory values will be 
generated. Association between GSTA1 enzymatic activity and busulfan metabolic rate (i.e. AUC, T1/2) 
will be determ ined using the Pearson correlation coefficient or, in case of a m onotone but non -linear 
relationship, by Spearm an’s rank correlation  coeffici ent.  To account for other determ
inants of 
metabolism, m ultiple linear regre ssion ana lysis will also  be perf ormed.  The relation
 b etween 
enzymatic activity and incidence of VOD will be assessing using logistic regression analysis.  Multiple 
logistic regression analysis may be attempted, depending on the frequency of VOD.  
 
 
The relationship between GST polymorphisms and VOD will be analyzed using chi-square or 
Fisher’s exact test. The association between polymorphisms and busulfan pharmacokinetics will 
be analyzed using a t-test or non-parametric Wilcoxon rank-sum test, as appropriate.   Finally, 
the pharmacogenomic determinants of drug efficacy will be assess using Cox (1972) 
multivariable regression analysis67 with time to disease progression or death as the outcome 
variable and genotype and busulfan pharmacokinetic parameters (AUC, etc.) as predictor 
variables.   
 
Estimated duration of accrual 
For the ph ase I tr ial, we estimate approximately 4-5 pa tients per month will b e accrued, so that the 
target of 30 subjects should be reached in 6-7 m
onths.  For the phase II study, w
e expect to accrue 
about 30 such patients per year.  A ssuming about 6- 10 patients from  the phase I study participate, a 
little over two years of recruitm ent and one year of  additional follow-up will be re quired to co mplete 
the phase II trial. 
 
15.0 DATA AND PROTOCOL MANAGEMENT 
 
15.1 
PROTOCOL COMPLIANCE Patients will be reviewed weekly during admission by the 
study investigators who will score the patient for standard endpoints. After discharge they 
will be reviewed at least once a month. 
 

 
31
15.2 
DATA ENTRY Data must be entered into the transplant database on a regular basis. 
Flowsheets will be kept for clinical and data collection purposes.  A brief explanation for 
required but missing data should be recorded as a comment. 
 
15.3 
ACCURACY OF DATA COLLECTION The Study Chairman will be the final arbiter of 
toxicity should a difference of opinion exist. 
15.4 REPORTING TO IBMTR AND NMDP  
As is customary in most transplant centers we plan to provide data to the International Bone 
Marrow Transplant Registry. We will also provide outcome data on unrelated donor 
recipients to NMDP as requested by that organization. Appropriate language will be 
included in the consent forms for this purpose. 
 
 
16.0 
CRITERIA FOR REMOVAL FROM PROTOCOL 
16.1 
At patient request. 
16.2 
Clinical progression. Such patients may be treated on other treatment protocols or 
at the investigator’s discretion. Such patients will continue to be monitored for 
survival and, may be asked to continue to provide specimens for studies of 
minimal residual disease and immune reconstitution as other treatments are 
recommended. 
 
17.0 REPORTING 
REQUIREMENTS 
 Any unexpected life-threatening and serious (grade 3 or 4) toxicity will be reported immediately 
to the Study Chairman.  The Chairman will be responsible for notifying the Surveillance 
Committee.  
Expected toxicities are those listed in the consent form and include regimen-related 
toxicities, myelosuppression, opportunistic infections such as CMV reactivation, or GVHD. 
These will not be routinely reported to the IRB even if they require admission.  
 
On the other hand, such toxicities will be monitored by the PI and the transplant team and 
reported regularly at the High Risk Protocol Committee of the cancer center. 
 
All deaths that are not due to disease recurrence will be reported to the IRB. 

 
32
 
 
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33
 
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24.  Sureda A, Perez dO, Garcia LJ, Odriozola J. High-dose busulfan and seizures. Ann.Intern.Med. 
1989;111:543-544. 
 
25.  Vassal G, Deroussent A, Hartmann O et al. Dose-dependent neurotoxicity of high-dose busulfan in 
children: a clinical and pharmacological study. Cancer Res. 1990;50:6203-6207. 
 
26.  Grochow LB. Busulfan disposition:  The role of therapeutic monitoring in bone marrow transplantation 
induction regimens. Semin.Oncol. 1993;20 Suppl. 4:18-25. 
 
27.  Bleyzac N, Souillet G, Magron P et al. Improved clinical outcome of paediatric bone marrow recipients 
using a test dose and Bayesian pharmacokinetic individualization of busulfan dosage regimens. Bone 
Marrow Transplant. 2001;28:743-751. 
 
28.  Kashyap A, Wingard J, Cagnoni P et al. Intravenous versus oral busulfan as part of a 
busulfan/cyclophosphamide preparative regimen for allogeneic hematopoietic stem cell transplantation: 
decreased incidence of hepatic venoocclusive disease (HVOD), HVOD-related mortality, and overall 100-
day mortality. Biol.Blood Marrow Transplant. 2002;8:493-500. 
 
29.  Andersson BS, Thall PF, Madden T et al. Busulfan systemic exposure relative to regimen-related toxicity 
and acute graft-versus-host disease: defining a therapeutic window for i.v. BuCy2 in chronic myelogenous 
leukemia. Biol.Blood Marrow Transplant. 2002;8:477-485. 
 
30.  McDonald GB, Slattery JT, Bouvier ME et al. Cyclophosphamide metabolism, liver toxicity, and mortality 
following hematopoietic stem cell transplantation. Blood 2003;101:2043-2048. 

 
34
 
31.  Hassan M, Ljungman P, Ringden O et al. The effect of busulphan on the pharmacokinetics of 
cyclophosphamide and its 4-hydroxy metabolite: time interval influence on therapeutic efficacy and 
therapy-related toxicity. Bone Marrow Transplant. 2000;25:915-924. 
 
32.  Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an 
alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of 
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33.  Bornhauser M, Storer B, Slattery JT et al. Conditioning with fludarabine and targeted busulfan for 
transplantation of allogeneic hematopoietic stem cells. Blood 2003;102:820-826. 
 
34.  Russell JA, Tran HT, Quinlan D et al. Once-daily intravenous busulfan given with fludarabine as 
conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical 
outcomes. Biol.Blood Marrow Transplant. 2002;8:468-476. 
 
35.  de Lima M, Couriel D, Thall PF et al. Once-daily intravenous busulfan and fludarabine: clinical and 
pharmacokinetic results of a myeloablative, reduced-toxicity conditioning regimen for allogeneic stem cell 
transplantation in AML and MDS. Blood 2004;104:857-864. 
 
36.  Slattery JT. Marrow transplantation for chronic myeloid leukemia: the influence of plasma busulfan levels 
on the outcome of transplantation. Blood. 1997;89:3055-3060. 
 
37.  Chattergoon DS, Saunders EF, Klein J et al. An improved limited sampling method for individualised 
busulphan dosing in bone marrow transplantation in children. Bone Marrow Transplant. 1997;20:347-354. 
 
38.  Hassan M, Fasth A, Gerritsen B et al. Busulphan kinetics and limited sampling model in children with 
leukemia and inherited disorders. Bone Marrow Transplant. 1996;18:843-850. 
 
39.  Slattery JT, Sanders JE, Buckner CD et al. Graft-rejection and toxicity following bone marrow 
transplantation in relation to busulfan pharmacokinetics. Bone Marrow Transplant. 1995;16:31-42. 
 
40.  Grochow LB. Parenteral busulfan: is therapeutic monitoring still warranted? Biol.Blood Marrow 
Transplant. 2002;8:465-467. 
 
41.  Vaughan WP, Carey D, Perry S, Westfall AO, Salzman DE. A limited sampling strategy for 
pharmacokinetic directed therapy with intravenous busulfan. Biol.Blood Marrow Transplant. 2002;8:619-
624. 
 
42.  van Besien K, Smith S., Odenike O et al. Alemtuzumab (Campath)-based GVHD prophylaxis leads to 
improved survival after allogeneic transplantation for patients with advanced hematologic malignancies 
[abstract]. Blood 2003;102:#2611. 
 
43.  Coles BF, Kadlubar FF. Detoxification of electrophilic compounds by glutathione S-transferase catalysis: 
determinants of individual response to chemical carcinogens and chemotherapeutic drugs? BioFactors 
2003;17:115-130. 
 
44.  Bredschneider M, Klein K, Murdter TE et al. Genetic polymorphisms of glutathione S-transferase A1, the 
major glutathione S-transferase in human liver: consequences for enzyme expression and busulfan 
conjugation. Clin.Pharmacol.Ther. 2002;71:479-487. 
 
45.  Dirven HA, van OB, van Bladeren PJ. Glutathione conjugation of alkylating cytostatic drugs with a 
nitrogen mustard group and the role of glutathione S-transferases. Chem.Res.Toxicol. 1996;9:351-360. 
 
46.  Czerwinski M, Gibbs JP, Slattery JT. Busulfan conjugation by glutathione S-transferases alpha, mu, and pi. 
Drug Metab Dispos. 1996;24:1015-1019. 

 
35
 
47.  Poonkuzhali B, Chandy M, Srivastava A, Dennison D, Krishnamoorthy R. Glutathione S-transferase 
activity influences busulfan pharmacokinetics in patients with beta thalassemia major undergoing bone 
marrow transplantation. Drug Metab Dispos. 2001;29:264-267. 
 
48.  Coles BF, Morel F, Rauch C et al. Effect of polymorphism in the human glutathione S-transferase A1 
promoter on hepatic GSTA1 and GSTA2 expression. Pharmacogenetics 2001;11:663-669. 
 
49.  DeLeve LD, Wang X. Role of oxidative stress and glutathione in busulfan toxicity in cultured murine 
hepatocytes. Pharmacology 2000;60:143-154. 
 
50.  Ritter CA, Sperker B, Grube M et al. Overexpression of glutathione S-transferase A1-1 in ECV 304 cells 
protects against busulfan mediated G2-arrest and induces tissue factor expression. Br.J.Pharmacol. 
2002;137:1100-1106. 
 
51.  Srivastava A, Poonkuzhali B, Shaji RV et al. Glutathione S-Transferase M1 Polymorphism- A Risk Factor 
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52.  Poonkuzhali B, Lapoumeroulie C, Shaji V. GSTM1 null genotype is associated with elevated GST  
expression in children with beta thalassemia major [abstract]. Blood 2002;100, Supp 1:abst 43473. 
 
53.  Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues: mechanisms of drug resistance and 
reversal strategies. Leukemia 2001;15:875-890. 
 
54.  Gati WP, Paterson AR, Belch AR et al. Es nucleoside transporter content of acute leukemia cells: role in 
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55.  Ritzel MW, Ng AM, Yao SY et al. Molecular identification and characterization of novel human and 
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57.  Hassan M, Oberg G, Bjorkholm M, Wallin I, Lindgren M. Influence of prophylactic anticonvulsant therapy 
on high-dose busulphan kinetics. Cancer Chemother.Pharmacol. 1993;33:181-186. 
 
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59.  Przepiorka D, Saliba R, Anderlini P et al. Chronic graft vs host disease after allogeneic stem cell 
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60.  McDonald GB, Hinds MS, Fisher LD et al. Veno-occlusive disease of the liver and multiorgan failure after 
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61.  Faries D. Practical modifications of the continual reassessment method for phase I cancer clinical trials. 
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36
 
65.  Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. Journal of the American 
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220. 
 
 
 
 
 
 

 
37
APPENDIX A: 
Treatment plan 
 
 
Day  
Clonazepam Fludarabine 
Busulfan 
Bu 
pharmacokinetics
Campath 
Test Dose 
- 
PO 
 
 
 
 
- 
PO 
 
0.5 mg/kg 
x 6 
 
- 
PO 
 
 
 
 
Treatment Doses 
-7 PO 
25 mg/m2  
 
20 mg 
-6 
PO  
25 mg/m2 
AUC based 
x 5 
20 mg 
-5 
PO  
25 mg/m2 AUC 
based 
 
20 mg 
-4 
PO  
25 mg/m2 
AUC based 
(x 5*) 
20 mg 
-3 
PO  
25 mg/m2 
AUC based 
x 5* 
20 mg 
-2 
PO 
   
 
-1 
 
   
 
0 
 
   
 
 
Comments: 
Busulfan administration has been associated with a low incidence of seizures and dilantin is 
routinely used for seizure prophylaxis. Dilantin use is discouraged in this protocol because 
exposure to dilantin can affect Busulfan metabolism.69 
We recommend benzodiazepines e.g clonazepam (klonopin ®) 1 mg PO TID 
Scheduling: 
Busulfan test dose: 
The test dose can be scheduled any day within 8 days prior to admission. Clonazepam will be 
started with an evening dose, the day prior to the test dose and two doses one day after test dose. 
Busulfan test-dose will be based on actual body weight and will be accompanied by extensive 
pharmacokinetic studies. This dose will be given as an outpatient. 
Patients will be admitted in the evening of day –8, to start fludarabine on day -7  
 
Fludarabine: 
Fludarabine dosing will be based on actual body weight. Fludarabine will be infused over 30 
minutes before busulfan treatment dose. 
 
Busulfan treatment doses: 
 
On each day of treatment, busulfan will be administered through a fresh infusion line at a steady 
rate over 3 hours. Infusion will start in the morning around 8 AM and will be completed around  
11 AM, allowing ample time for pharmacokinetic sampling in the afternoon.  
*Pharmacokinetic studies will be performed on the day 1 dose and again on the day 4 dose. For 
logistical reasons (week-end, holidays), day 3 pharmacokinetics may be performed instead of 
day 4. 
 
 

 
38
Campath 
All patients will receive premedication for Campath as per current standards at the UC transplant 
unit. 
18.1.1 Dose escalation schedule (see also statistical plan)  
The targed AUC of busulfan will be escalated and de-escalated in an up and down design to 
achieve a dose where less than 4 of 12 patients have a DLT.  
 
Target AUC’s are the following: 
Dose level 0: 4800 mcgr.min/L.dose 
Dose level 1: 5800 mcgr.min/L.dose 
Dose level 2: 6800 mcgr.min/L.dose 
Dose level 3: 7800 mcgr.min/L.dose 
Dose level 4: 8800 mcgr.min/L.dose 
Dose level 5: 9800 mcgr.min/L.dose 
 
We do not anticipate further dose-escalation beyond dose level 5. If such dose-escalation were to 
be considered, we would resubmit an amended protocol to the IRB. 
 
18.1.2 Bone marrow/stem cel infusion 
On day 0 the stem cell product will be infused according to BMT unit policy. 
Bone marrow, PBSC will not be further processed except in the case of ABO-incompatibility. In 
such instances, red blood cells will be removed from bone marrow products or from PBSC 
products containing excessive amounts of RBC (as per transplant policy). 
All stem cell products procured through the NMDP will be done so in strict compliance with the 
protocols, policies, and procedures established by the NMDP.  
 
 
18.1.3 GVHD prophylaxis  
 Tacrolimus 0.03 mg/kg/day IV CI over 24 hr from 4 PM day -2 until engraftment or when 
patient is able to take PO, then tacrolimus 0.09 mg/kg PO in 2 divided doses.  Tacrolimus should 
be given at full dose to maintain levels of 5-15 ng/mL through day 100. Thereafter tacrolimus 
will be tapered by 20% every week unless the patient has developed GVHD. 
 
18.1.4 Supportive care:  
Infection prophylaxis and supportive care will be as BMT unit policy.  No routine growth factor 
support will be administered.Growth factor support will be considered in case of delayed 
engraftment (ANC < 0.5x109/L on day 12) 
 
18.1.5 Back-up bone marrow harvest (only for recipients of mismatched or 
unrelated donor transplants) 
For patients transplanted in remission, a backup bone marrow or peripheral blood stem cell 
collection will be considered within two weeks prior to hospital admission as long as the overall 
marrow cellularity is greater than 30%. All backup harvests will be cryopreserved after 
processing for buffy coat without any further manipulation. The backup stem cells will be given 
if deemed clinically necessary in the event of failure to engraft by day +35, graft rejection after 
day +35, or graft failure after day +35. 
 

 
39
APPENDIX B 
 
GVHD GRADING CRITERIA  
 
CLINICAL GRADING OF ACUTE GVHD  
(Thomas et al., NEJM, 229:895, 1975) 
 
 
Grade 
Degree of Organ Involvement                    
____________________________________________________________ 
I 
 
+ to ++ skin rash; no gut involvement; no liver involvement; no 
decrease in clinical performance 
 
II 
 
+ top +++ skin rash; + gut involvement or + liver involvement (or 
both); mild decrease in clinical performance 
 
III 
 
++ to +++ skin rash; ++ to +++ gut involvement or ++ to ++++ liver 
involvement (or both); marked decrease in clinical performance 
 
IV 
 
Similar to grade III with ++ to ++++ organ involvement and extreme 
decrease in clinical  
 
performance 
_____________________________________________________________ 
 
Stage   
Skin                    
Liver             
Intestinal Tract 
______________________________________________________________ 
 
+    
Maculopapular rash    Bilirubin 
 
>500 ml 
 
   
<25% to body surface   2-3 mg/100 ml 
diarrhea/day  
 
 
 
 
 
++    
Maculopapular rash    Bilirubin 
 
>1000 ml 
 
   
25-50% body surface    3-6 mg/100 ml 
diarrhea/day  
 
 
 
 
 
+++ 
   
Generalized   
     
Bilirubin 
 
>1500 ml 
 
   
erythroderma  
    
6-15 mg/100 ml 
diarrhea/day  
 
++++ 
   
Generalized   
Bilirubin 
 
Severe abdominal  
 
   
erythroderma with      
>15 mg/100 ml 
pain, with or  
 
   
bullous formation 
 
 
 
 
without ileus 
 
   
and desquamation 
 
 

 
40
CLINICAL GRADING OF CHRONIC GVHD 
 
(Shulman et al.  Am J Med 69:204, 1980) 
 
Extensive - multiorgan involvement clinically 
Limited - only skin involvement clinically 
Subclinical - only histologic evidence 
 
 
 
 
 
 

 
41
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