Pediatr Blood Cancer 2010;55:254–259
Phase I Study of Bortezomib Combined With Chemotherapy in Children
With Relapsed Childhood Acute Lymphoblastic Leukemia (ALL): A Report
From the Therapeutic Advances in Childhood Leukemia (TACL) Consortium
Yoav Messinger, MD,1* Paul Gaynon, MD,2Elizabeth Raetz, MD,3Raymond Hutchinson, MS, MD,4
Steven DuBois, MD,5Julia Glade-Bender, MD,6Richard Sposto, PhD,7Jeannette van der Giessen, BA,7
Elena Eckroth, CCRC,7and Bruce C. Bostrom, MD8
Despite progress inchildhoodALL, outcomesremain poor after
from diagnosis) have fared worse with a 5-year survival of about
achieving complete remission (CR) after a single treatment block
remission reinduction rate of about 70% after one treatment block
early relapsed ALL (less than 18 months from diagnosis) had low
Advances in Childhood Leukemia (TACL) institutions found a
remission reinduction rate of 78% for very early and 86% CR for
early first marrow relapse . Remissions were more difficult to
achieve in children with multiple recurrences. The TACL group
observed low remission rates of 44%, 27%, and 12% for third,
fourth, and further therapeutic attempts, respectively . In that
study disease-free survival (DFS) of patients in CR3 or above was
only 15% at 5 years, indicating the poor outcome of multiple
marrow relapsed ALL has been documented over the past 20 years
 and outcomes remain poor, new therapeutic strategies for
relapsed childhood leukemia are urgently needed.
in this population based on promising preclinical activity and
success in other hematopoietic malignancies. Bortezomib is a
selective inhibitor of the ubiquitin proteasome pathway (UPP),
which is an essential pathway for the degradation of most
proteins in eukaryotic cells . Important regulatory proteins
affected by inhibition of the UPP system include NF-kB, p53, Bax,
p27, and p21 . Proteasome inhibition may sensitize malignant
hematologic cells to apoptosis induced by both radiation and
chemotherapy [7–9]. Bortezomib has received FDA approval
for use in adults with multiple myeloma and relapsed NHL .
Preclinical studies did show some activity of single agent
bortezomib in ALL . Unfortunately, clinical studies showed
or children  with acute leukemia.
Bortezomib may have enhanced anti-leukemic activity when
used in combination with chemotherapy. Horton et al. 
have shown that bortezomib and dexamethasone have synergistic
anti-leukemia interactions in vitro, and bortezomib had additive
anti-leukemia effects when combined with asparaginase, vincris-
tine, doxorubicin, or cytosine arabinoside. A multiply relapsed
child with ALL had transient clinical response to bortezomib and
Background. Outcomes remain poor for children after relapse of
acute lymphoblastic leukemia (ALL), especially after early marrow
relapse. Bortezomib is a proteasome inhibitor with in vitro synergy
with corticosteroids and clinical activity in human lymphoid
malignancies. Procedure. This is a Phase I study of escalating
doses bortezomib administered days 1, 4, 8, and 11, added to 4-drug
induction chemotherapy with vincristine, dexamethasone, pegylated
L-asparaginase, and doxorubicin (VXLD) in children with relapsed
ALL. Results. Ten patients were enrolled, five in first marrow relapse,
and five in second relapse. Four patients were enrolled at dose level
1 (bortezomib 1mg/m2). One patient was not evaluable for toxicity
because of omitted dexamethasone doses. No dose-limiting toxicity
(DLT) was observed. Six patients were enrolled at dose level 2
(bortezomib 1.3mg/m2). One patient had dose-limiting hypophos-
phatemia and rhabdomyolysis after 1 dose of bortezomib, and died
from a diffuse zygomyces infection on day 17. Five additional
patients were enrolled with no subsequent DLTs. As planned, no
further dose escalation was pursued. The regimen had predictable
toxicity related to the chemotherapy drugs. Two patients had mild
peripheral neuropathy (grades 1 and 2). Six of nine evaluable
patients (67%) achieved a complete response (CR), and one had a
bone marrow CR with persistent central nervous system leukemia.
Conclusions. The combination of bortezomib (1.3mg/m2) with
VXLD is active with acceptable toxicity in pretreated pediatric
patients with relapsed ALL. We are expanding the 1.3mg/m2
cohort for a phase II estimate of response. Study registered at
Pediatr Blood Cancer 2010;55:254–259.
? 2010 Wiley-Liss, Inc.
ALL relapse; bortezomib; chemotherapy; phase I study
? 2010 Wiley-Liss, Inc.
Published online 20 April 2010 in Wiley InterScience
1Pediatric Hematology and Oncology, Children’s Hospitals and Clinics
of Minnesota, St. Paul, Minnesota;
California;3Department of Pediatrics, New York University School
of Medicine, New York, New York;4University of Michigan Medical
School, Ann Arbor, Michigan;
School of Medicine, San Francisco, California;6Pediatric Oncology,
Columbia University, New York, New York;7Children’s Center for
Cancer and Blood Diseases, Children’s Hospital Los Angeles, Los
Angeles, California;8Pediatric Hematology and Oncology, Children’s
Hospitals and Clinics of Minnesota, Minneapolis, Minnesota
2Division of Hematology/
5Department of Pediatrics, UCSF
Conflict of interest: Paul Gaynon, MD: honoraria from Enzon, Allos,
and Sanofi Aventis, Chair of data monitoring committee (dasatinib) for
Bristol Meyers. No one has any conflict of interest with Millennium
Pharmaceuticals, or reports any other conflict of interest. Millennium
Pharmaceuticals did not sponsor this study, or provide funds or drug
Children’s Hospitals and Clinics of Minnesota, 345 N. Smith Avenue,
St. Paul, MN 55102. E-mail: email@example.com
Received 6 November 2009; Accepted 6 January 2010
dexamethasone . In a phase I adult acute myeloid leukemia
(AML), Attar et al.  have shown that the combination of
bortezomib with idarubicin and cytosine arabinoside resulted in
61% CR’s and an additional 10% with CR’s butincomplete platelet
Based on these studies, we added bortezomib to a standard
chemotherapy platform of vincristine, dexamethasone, pegylated
L-asparaginase (PEG-asparaginase), and doxorubicin (VXLD)
platform. In a phase I/II study, we sought to determine whether
patients. The phase I part of this study is reported here.
PATIENTS AND METHODS
This study was approved by the Institutional Review Boards
(IRBs) of all participating TACL centers. An independent Data
Safety Monitoring Committee at Children’s Hospital of Los
Angeles monitored study progress.
Individual and/or parental informed consent was obtained from
all subjects as per local and federal requirements. Eligible patients
at least one prior treatment attempt, with no limit placed on the
number of prior treatment regimens. Patients were eligible after
actively for graft-versus-host-disease.
of age, or 10 years of age or younger, respectively, were required at
study entry. Allergy to asparaginase products, boron or mannitol,
prior exposure to ?350mg/m2of anthracycline (in doxorubicin
30% excluded participation. Other exclusion criteria were: serum
bilirubin >1.5 times the institutional ULN; ALT >4 times ULN;
history of pancreatitis; serum amylase or lipase >2 times ULN; or
active uncontrolled infection. As neuropathy has been described
both for bortezomib and vincristine, patients with pre-existing
?grade 2 motor or sensory neuropathy were also excluded.
Treatment and Dose Escalation
The treatment regimen is shown in Table I. Only 1 course of
therapy was planned. Bortezomib dose escalation included dose
levels of 1mg/m2/dose (starting dose level) and 1.3mg/m2/dose
(maximum planned dose level). De-escalation of bortezomib to
0.7mg/m2/dose was planned if dose-limiting toxicity (DLT) was
observed at the starting dose level. No dose escalation of the
other chemotherapy agents was planned. Originally, CNS therapy
of CNS status. After four patients were enrolled and a patient with
CNS involvement did not clear the CSF, weekly triple intrathecal
chemotherapy was introduced for patients with CNS disease.
Patients were enrolled in groups of three patients, with dose
escalation following a standard 3þ3 patient cohort escalation
design . Briefly, 0/3 DLTs would allow escalation, 1/3 DLTs
DLTs required de-escalation, and the maximum tolerated dose
DLTs was observed. Dose escalation was based on DLTs occurring
during the single planned course of therapy.
Toxicity was graded using the CTCAE criteria, version 3.0
(http://ctep.cancer.gov). DLT was defined as any of the following
toxicities deemed by the investigator as possibly, probably, or
definitely attributable to bortezomib: ?grade 3 sensory neuropathy
or neuropathic pain; marrow hypoplasia (less than 10% cellularity)
which continued 6 weeks from the start of the course; grade 4 non-
hematologic toxicity excluding infection (septic shock, typhlitis),
fever and neutropenia, fatigue, electrolyte abnormalities, hyper- or
hypoglycemia, nausea or vomiting, or AST, ALT, or bilirubin
elevations that returned to grade 1 by the time of blood count
recovery following completion of the course.
could continue with the remainder of the backbone therapy
excluding bortezomib. PEG-asparaginase was to be discontinued
Pediatr Blood Cancer DOI 10.1002/pbc
TABLE I. Treatment Regimen
Bortezomib and Chemotherapy in Relapsed ALL255
for anaphylaxis or systemic allergic reactions, symptomatic
pancreatitis, significant bleeding, or significant thrombosis. No
modification of PEG-asparaginase or dexamethasone for hyper-
glycemia was planned.
Bone marrow aspirate/biopsy and CBC to assess response were
done on day 29. A bone marrow procedure was not required if the
patient has an absolute blast count (ABC) greater than or equal to
2,500/ml in the peripheral blood on day 29. If the marrow was
hypoplastic and peripheral blood counts had not recovered to
absolute neutrophil count (ANC) >750/ml and a platelet count
>75,000/ml, a repeat marrow aspiration and complete blood count
were required weekly until recovery or progression. If the patient
had ?5% lymphoblasts in the bone marrow (M1 marrow) but did
was repeated weekly until count recovery or relapse.
The following definitions of response were used: CR—M1
(<5%blasts) bone marrowwithno evidence ofcirculating blastsor
extramedullary disease and with recovery of peripheral counts
(absolute neutrophil count (ANC) >750/ml and platelet count
ANC >750/ml, but insufficient recovery of platelets (<75,000/ml);
sites of extramedullary disease, and with recovery of absolute
neutrophil counts (ANC >750/ml); stable disease (SD), does not
satisfy the criterion for PD, or has recovery of ANC >750/ml but
fails to qualify for CR, CRp, or PR; progressive disease (PD), an
increase of at least 25% in the absolute number of circulating
or other laboratory or clinical evidence of PD, with or without
recoveryofANCorplatelets.Minimal residualdisease (MRD) was
not evaluated as a part of this study.
Patients and Dose Escalation
A total of 10 patients were enrolled in the phase I portion of this
one patient agewas 9.8 years (range 3.3–16.4 years). Nine patients
hadB-precursor ALL and one patient had T-cell ALL. Five patients
were treated in first relapse, (very early relapse <18 months, one
patient; early relapse 18–36 months, one patient; late relapse
>36 months, three patients). Five patients were in second relapse.
None of the patients had primary refractory disease, and none had
prior stem–cell transplantation.
A total offour patients were treated on dose-level 1 (bortezomib
didnot receive the correct doses of dexamethasone, was considered
incompletely evaluable for toxicity and not evaluable for response.
This patient was removed from further protocol therapy after day 8
and replaced to ensure a total of three fully evaluable patients. No
DLTwas seen, so escalation to dose level 2 was initiated.
A total of six patients were then enrolled into dose level 2
(bortezomib 0.3mg/m2/dose) (Table II). The first patient on this
dose level suffered DLT immediately after the first dose of
bortezomib. Based on the rule that 1/3 DLTs required expanding
Pediatr Blood Cancer DOI 10.1002/pbc
TABLE II. Clinical Data
Number of prior
Age at study
Off therapy—day 8
Off therapy due to
system.aLength of first remission of patients treated at first relapse: Very early <18 months, Early 18–36, Late relapse >36 months.
256 Messinger et al.
to six patients, an additional five patients were enrolled to that dose
level. No further DLT was seen and dose level 2 was deemed
tolerable. Since no further escalation of bortezomib was planned,
the phase I portion of the study was completed and dose level 2
(1.3mg/m2/dose) was selected for further Phase II evaluation.
Table III. Patient T0015 was enrolled on the study at dose level 2
(1.3mg/m2/day). The patient had received the first dose of
vincristine, bortezomib, and doxorubicin, but had not yet received
dexamethasone. Seventy minutes after the bortezomib she devel-
ness. She experienced grade 4 hypophosphatemia, and grade 3
myositis (rhabdomyolysis) with elevated creatine phosphokinase,
aldolase, and myoglobinuria. The patient did not receive additional
bortezomib. The patient recovered from this toxicity with the
exception of continued grade 1 hypophosphatemia. This patient
suffered hypophosphatemia duringher original diagnosisand again
regimen-related. However, this constellation of symptoms was
determined to be a DLT. The patient continued with the chemo-
therapy backbone of the regimen (excluding bortezomib) and
DLT or regimen-associated mortality was seen on this part of the
As expected all patients suffered hematologic grade 4 toxicity.
patients achieved platelet >75,000/ml at median day 34 (range 27–
of the regimen-relatedtoxicity are shownin
42) and ANC >750/ml at median day 41 (range 28–43). Infectious
complications included three episodes of bacteremia and one
systemic fungal infection (the only death on this regimen). Notable
also were electrolyte disturbances including hyponatremia (50%),
which has been described after bortezomib and vincristine. In one
patient, SIADH may have been the cause of the hyponatremia.
Hypokalemia and dehydration were also observed. Hypophospha-
temia grade 3-4 was seen in two patients but was symptomatic only
in the one patient described above, which was associated with
rhabdomyolysis and subsequent mental status changes. Significant
neurologic events included: confusion and myositis secondary to
rhabdomyolysis from hypophosphatemia; depressed sensorium
secondary to dehydration that recovered after fluids; cerebral
ischemia from small acute cortical infarctions secondary to PEG-
asparaginase, which was then discontinued; and syncope that
resolved with no intervention. Interestingly, two patients had mild
peripheral neuropathy (grades 1 and 2), requiring no intervention,
such as therapy interruption or dose modification. There was no
discernible difference in the distribution of toxicity reporting
between dose levels 1 and 2.
Nine patients are evaluable for response. Six patients achieveda
CR (67%), and an additional one patient achieved a CR in the bone
marrow but had persistent CNS leukemia (patient did not receive
weekly intrathecal triple chemotherapy, which was introduced
thereafter). Therefore, 7/9 (78%) achieved a bone marrow CR. One
patient achieved SD and another patient died on day 17 before
response could be evaluated. Of patients in first relapse, 3/4 (75%)
Pediatr Blood Cancer DOI 10.1002/pbc
TABLE III. Grades 3–5 Toxicity Data in the 10 Patients Evaluable for Toxicity
Group ToxicityGrade 3 Grade 4 Grade 5Total
Implant site Infection
Infection with neutropenia
Confusion and myositis
Prolonged QT interval
Bortezomib and Chemotherapy in Relapsed ALL 257
had bone marrow CR, and of patients in second relapse 4/5 (80%)
to proceed to the phase II part of this study, which is accruing
patients at this time.
This phase I study in childhood ALL was designed to evaluate
whether combining bortezomib with a previously active chemo-
therapy backbone is tolerable. It demonstrated that a standard dose
of bortezomib 1.3mg/m2given on days 1, 4, 8, and 11 can be safely
combined with an intensive 4-drug reinduction regimen in children
withrelapsed ALL. Sincethe phaseIpediatric studyof singleagent
further dose escalation in the combination trial. Similarly, in adults
with AML, bortezomib 1.5mg/m2on the same schedule was safely
combined with idarubicin and cytosine arabinoside .
There was no prolonged bone marrow suppression from this
regimen, even though thrombocytopenia is well recognized after
bortezomib administration. As previously described , infections
are common with a similar chemotherapy backbone and can lead
to death from fungal infection. Although both bortezomib and
vincristine can induce peripheral neuropathy only two patients
suffered grade 1-2 transient peripheral neuropathy. However, four
significant neurologic events (confusion, depressed sensorium,
chemotherapy agents. Patients on the phase II portion of the study
receive close monitoring for neurologic toxicity. Pulmonary
toxicity, described after bortezomib use in adults , was not
observed in this study.
Interestingly, three of four patients in first relapse achieved a
complete response (CR), but more significantly, four of five
in second relapse achieved complete bone marrow response
(Table IV). This is a higher response rate than the 44% CR
reinduction rates in an expanded number of patients in second or
above relapse is the primary objective of the phase II part of the
study. Although early, this level of response is impressive because
very limited single agent bortezomib activity was seen in leukemia
in adults  and children . If confirmed in the phase II part of
this study, it may corroborate the in vitro synergistic activity with
dexamethasone  as well as the clinical activity previously
observed in one patient .
in second relapse reported here. Additionally, the data reported
here were used by the COG to initiate another phase II study of
The mechanism by which proteasome inhibition leads to
increased sensitivity to chemotherapeutic agents is not clear. It
was previously shown that inhibition of NF-kB, which is
constitutively elevated in leukemia cells, enhances the sensitivity
of AML cells to chemotherapeutic agents . In contrast,
inhibition of NF-kB expression did not enhance chemosensitivity
of ALL cell-lines . Whether bortezomib-induced inhibition
of NF-kB is the primary mechanism inducing leukemia cell death
in vivo is uncertain. In the single agent trial of bortezomib, Horton
et al.  observed increased NF-kB expression 6–12hr after
bortezomib exposure, followed by a 4–6-fold decrease in NF-kB
expression 24hr after bortezomib exposure. In contrast, Attar et al.
reported in vivo reduction of NF-kB expression within 1hr after
bortezomib . Bortezomib was shown to down-regulate IkB
expression and trigger NF-kB activation in multiple myeloma,
raising questions about the prevailing model that bortezomib is an
NF-kB inhibitor . Others describe that proteasome inhibition
results in accumulation of Bax and Bim pro-apoptotic proteins,
which sensitize leukemia cells to anthracycline-induced apoptosis
. Elucidation of the specific pathways involved may allow for
additional targeted agents to be used in combination with
used in the current regimen.
In summary, this phase I study of bortezomib given in standard
twice-weekly doses of 1.3mg/m2/dose, combined with vincristine,
dexamethasone, PEG-asparaginase, and doxorubicin is tolerable
and shows promising activity in relapsed childhood ALL.
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TABLE IV. Response
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Death or no
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Bortezomib and Chemotherapy in Relapsed ALL259