Received: 15 March 2017
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Revised: 23 March 2017
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Accepted: 27 March 2017
DOI: 10.1002/ajh.24746
A JH
RESEARCH ARTICLE
An exploratory clinical trial of bortezomib in patients with lower risk myelodysplastic syndromes May Daher1
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Juliana Elisa Hidalgo Lopez2 | Jasleen K. Randhawa3 |
Kausar Jabeen Jabbar2 | Yue Wei3 | Naveen Pemmaraju3 Tapan Kadia3 | Marina Konopleva3 | Hagop M. Kantarjian3
Gautam Borthakur3 |
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Katherine Hearn3 |
Zeev Estrov3 | Steven Reyes2 | Carlos E. Bueso-Ramos2 | Guillermo Garcia-Manero3 1 Division of Cancer Medicine, The University of Texas M.D. Anderson Cancer Center, Texas, USA; 2Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Texas, USA; 3 Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Texas, USA
Abstract Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis and an increased risk of transformation. Few effective therapies are available for lower risk MDS patients, especially after the failure of hypomethylating agents. MDS progenitor cells are dependent on the nuclear factor-jB (NF-jB) for survival, which makes it an attractive therapeutic target. As a proteosomal inhibitor, bortezomib is thought to have inhibitory activity against NF-jB. We designed a
Correspondence Guillermo Garcia-Manero, MD, University of Texas, MD Anderson Cancer Center, Box 428, 1515 Holcombe Blvd, Houston, TX 77030, USA. Email:
[email protected]
proof-of-principle study of subcutaneous (SC) bortezomib in lower risk MDS patients with evi-
Funding information Millennium Pharmaceuticals; the MD Anderson Cancer Center Support Grant, Grant/Award Number: P30 CA16672; the Dr. Kenneth B. McCredie Chair in Clinical Leukemia Research endowment; the Edward P. Evans Foundation; the Fundacion Ramon Areces; the Cancer Prevention & Research Institute of Texas (CPRIT) award, Grant/Award Number: RP141500; to MD Anderson’s MDS/AML Moon Shot Program
excess toxicity. Three patients out of the 15 (20%) had evidence of response with hematologic
dence of NF-jB activation in their bone marrow. Fifteen patients were treated, their median age was 71 (range 56–87), 33% were low and 67% int-1 by IPSS, median number of prior therapies was 2, all patients were transfusion dependent. Baseline median pp65 percentage was 31% and 11 patients had evidence of ring sideroblasts (RS). SC bortezomib was safe, well tolerated with no improvement (HI-E). Bortezomib caused a decrease in pp65 levels in 7 out of 13 evaluable patients (54%, P 5 .025). Of interest, unexpectedly, we observed a significant decrease in RS in 7 out of 10 (70%) evaluable patients during treatment. In conclusion, this study suggests that NF-jB activation, measured by pp65 levels, may be a useful biomarker in MDS. Bortezomib is safe in this patient population but has modest clinical activity. The role of the proteasome in the genesis of RS needs further study.
1 | INTRODUCTION
(TLRs) are a family of pattern-recognition receptors that play a pivotal role in the innate immunity. These receptors activate a common signaling
Myelodysplastic syndromes (MDS) represent a spectrum of clonal hema-
pathway through adaptor protein MyD88, culminating in the activation
topoietic disorders that affect the myeloid lineage, characterized by inef-
of nuclear factor-kB (NF-jB).6,7 Recent studies in MDS demonstrated a
fective hematopoiesis and an increased risk of transformation to acute
constitutive activation of genes that involve a variety of different steps
1
myeloid leukemia (AML). Patients with MDS are stratified into lower or 2,3
higher-risk categories based on IPSS or IPSS-R classifications.
For a
of this pathway.8–11 This evidence implicates NF-jB in the pathogenesis of MDS, and, hence, makes it a potential therapeutic target.
majority of lower risk (low or int-1) MDS patients, the standard of care
The NF-jB transcription factor family is composed of five proteins
remains suboptimal with few effective therapeutic alternatives, particu-
(p50, p52, p65, c-Rel, and RelB) that play a role in apoptosis regulation,
larly for patients previously treated with a hypomethylating agent
cell proliferation, survival, and metastasis.12 It is, therefore, not surpris-
4
(HMA). Novel treatment approaches are needed for these patients.
ing that it has been linked to the pathogenesis of multiple solid and
Accumulating evidence from recent clinical and molecular studies
hematologic malignancies.12–15 Within the canonical pathway, mem-
supports a key role for deregulation of the innate immune system and
bers of the NF-jB family form dimers (most commonly heterodimers
5
inflammatory signaling in the pathogenesis of MDS. Toll-like receptors
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of p65 with p50) which are retained in the cytoplasm through
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Am J Hematol. 2017;92:674–682.
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interactions with inhibitory molecules of the inhibitor of the NF-jB
for MDS were allowed to participate in the study. Patients were
(IjB) family.12,16 Activating stimuli (including cytokines and various
required to have adequate liver and renal function and were required
stress signals) lead to phosphorylation of the IjB molecules by IjB
to practice effective methods of contraception if not surgically sterile.
kinases (IKKs) and their degradation via the ubiquitin-proteasome path-
Female patients of childbearing age were required to have a negative
17
way.
Consequently, the NF-jB dimers are liberated and are then free
pregnancy test within 72 h of treatment.
to translocate to the nucleus and activate their target genes.18 Phos-
Patients were excluded if they had any severe concurrent disease
phorylation of p65 is required for nuclear translocation and transcrip-
or condition that would make them inappropriate for study participa-
tional activation of target genes involved in proliferation, stress
tion, including a myocardial infarction within 6 months of enrollment,
19,20
response and cytokine production.
Therefore, phosphorylated p65
(pp65) may be used as a clinical surrogate for NF-jB activation.
New York Heart Association (NYHA) Class III or IV heart failure, uncontrolled angina, uncontrolled ventricular arrhythmias, or electrocardio-
The NF-jB subunit RelA/p65 has also been described as an impor-
graphic evidence of active ischemia or active conduction system
tant factor in hematopoiesis through the regulation of hematopoietic
abnormalities. Patients with other concomitant malignancies were
stem cell function and lineage commitment. In addition, p65 has been
excluded, with the exception of a completely resected basal cell carci-
shown to play a potential role in the regulation of cell differentiation
noma or squamous cell carcinoma of the skin, an in situ malignancy, or
via up-regulation of additional NF-jB subunits; suggesting that, p65
low-risk prostate cancer after curative therapy. Other exclusion criteria
21,22
may influence specific lineage-committed cell types.
Bortezomib is a reversible selective inhibitor of the 26S proteoso-
included confirmed pregnancy or lactation, hypersensitivity to bortezomib, boron or mannitol and peripheral neuropathy grade 2 or higher.
mal subunit with previously described inhibitory activity against NFjB.23–25 In preclinical and clinical studies, bortezomib was shown to induce apoptosis in malignant cells of hematopoietic lineage via inhibition of the NF-jB pathway.26,27 Leukemic stem cells are highly dependent on NF-jB for survival28,29; MDS progenitors are also highly dependent on NF-jB activation for survival.10,30 In vitro studies of MDS cells have shown that NF-jB inhibition leads to an autophagic stress response followed by apoptotic cell death.31 In clinical studies, bortezomib has shown modest activity in myeloid neoplasms, both as a
2.2 | Correlative studies We analyzed phosphorylated-p65 (pp65) level at baseline on day 21 of cycles 1 and 3 and then in subsequent marrows as clinically indicated. Bone marrow aspirate smears were fixed for 30 minutes in fresh absolute methanol at 2–88C. After fixation, the thick area of the BM smears was scraped off the slide while wet with methanol and allowed to air dry for 10 min at room temperature. Following incubation for 20 min in 31 TBST (Tris-Buffer Saline Tween 20, Dako North America
single agent and in combination with other cytotoxic agents.32–36 Com-
Carpinteria, CA), immunofluorescence was assessed by an indirect
parison between subcutaneous (SC) and intravenous administration of
method in a Dako autostainer Link 48 at room temperature. Smears
bortezomib showed that SC administration offers similar efficacy, with
were stained using 1:20 of 300 ul of the rabbit polyclonal antibody
improved safety profile, especially with a significantly lower risk of
reactive with NF-kB p65 [p-NFқB p65 (Ser276) antibody, Santa Cruz
peripheral neuropathy.37,38
Biotechnology, TX, USA] for 30 min followed by 1 wash with1X TBST.
As a proof of concept, we designed this study using SC bortezomib
This assay was performed in the Department of Hematopathology fol-
in patients with low or intermediate-1 risk MDS who had evidence of
lowing Clinical Laboratory Improvement Amendments regulations at
NF-jB activation. Our objectives were primarily to determine the
The University of Texas MD Anderson Cancer Center. The antibody–
effect of SC bortezomib on modulation of NF-jB activity and second-
antigen complexes were detected by incubation for 30 min using 1:5
arily to determine the clinical activity, safety, and tolerability of SC bor-
of 300 uL of FITC–conjugated secondary goat anti-rabbit immunoglob-
tezomib in these patients.
ulin G, (Fab2) antibody (FITC, Supertechs, MD) and 2 washings of 31 TBST. Slides were embedded in antifade Vectashield mounting medium
2 | PATIENTS AND METHODS
with Propidium iodide (PI); (Vectashield mounting medium with PI, Vectashield, CA) cover slipped, and analyzed with a conventional fluores-
2.1 | Eligibility criteria Eligible patients had a confirmed diagnosis of MDS according to WHO or FAB criteria,39–41 and were classified by the IPSS as low or int-1 risk MDS within 28 days of the first dose of treatment. Patients were required to have a presence of the phosphorylated p65 NF-jB component in at least 5% of their bone marrow (BM) cells. Eligible patients were at least 18 years of age, had an Eastern Cooperative Oncology
cence microscope Olympus BX53 and DP73 digital camera with CellSens program (Olympus BX53 and DP73 digital camera with CellSens program, Olympus America, MA).42 Positive results were defined as nuclear fluorescence phosphorylated p65 NF-jB in at least 5% of BM cells.
2.3 | Cytogenetic studies
Group (ECOG) performance status of 2 or less, and must have received
Conventional chromosomal analysis was performed on G-banded
at least one prior therapy for MDS. Any prior systemic treatment had
metaphases that were prepared from unstimulated 24 and 48-h BM
to be completed 14 days prior to the study start date, and any prior
aspirate cultures using standard techniques. The results were reported
radiation therapy had to be completed within 3 weeks prior to enroll-
using the 2016 International System for Human Cytogenetics Nomen-
ment. Patients who had received a prior allogeneic stem cell transplant
clature as described previously.43,44
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2.4 | Molecular studies
2.6 | Response and toxicity assessment
We performed mutation analysis using a 28-gene panel as previously
Adverse events were graded and reported according to the National
described.45–48 Briefly, genomic DNA was extracted from BM aspirates
Cancer Institute Common Terminology Criteria for Adverse Events ver-
or peripheral blood. Amplicon-based next-generation sequencing (NGS)
sion 4.0. All patients were considered evaluable for toxicity from the
targeting the entire coding regions of a panel of 28 genes associated
time of their first treatment with bortezomib. All treated patients were
with myeloid neoplasms was performed using a MiSeq platform (Illu-
evaluated for response. Response criteria were assessed according to
mina, San Diego, CA). The genes analyzed included ABL1, ASXL1, BRAF,
the modified IWG criteria.52,53 Responses included complete remission
DNA methyltransferase (DNMT3A), EGFR, EZH2, FLT3, GATA1, GATA2,
(defined as BM 5% myeloblasts with normal maturation of all cell lines
HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88,
and peripheral blood Hgb 11 g/dL, platelets 100 3 109/L, neutrophils
NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, and WT1. A
1.0 3 109/L, blasts 0%), partial remission (defined as BM blasts
sequencing library was prepared using 250 ng of DNA template. Equal
decreased by 50% over pretreatment but still > 5%), marrow complete
quantities of DNA from purified sequencing libraries were used for Tru-
remission (defined as BM 5% myeloblasts and decrease by 50% over
Seq paired-end sequencing on the MiSeq sequencer using the MiSeq
pretreatment) and any hematological improvement achieved at any
Reagent Kit v2 (500 cycles). Variant calling was performed with Illu-
time during the duration of the therapy. Event-free survival (EFS) was
mina MiSeq Reporter Software using human genome build 19 (hg 19)
defined as the time between the start of therapy and the date of lack of
as a reference. For clinical reporting, a minimum sequencing coverage
response, loss of response, transformation to AML, or death, whichever
of 3250 (bidirectional true paired-end sequencing) was required. The
occurred first. OS was defined as the time between the start of therapy
analytical sensitivity was established at 5% mutant reads in a back-
and death. Patients who were alive at the last follow-up date were cen-
ground of wild-type reads. CEBPA was performed using PCR followed
sored in survival analyses. The protocol is available in the Supporting
by Sanger sequencing. Internal tandem duplications of FLT3 gene and
Information material section for detailed definitions of response criteria.
NPM1 mutation (exon12) were assessed using PCR followed by capillary electrophoresis (all of these methods have been previously described).45,49,50 PCR-based DNA sequencing was then used to iden-
2.7 | Statistical considerations
tify: RAS mutation (codons 12, 13, and 61 of the KRAS and NRAS),
The primary objective of the study was to investigate the effect of SC
IDH1R132 mutation (codon 87 to 138 in exon 4), IDH2 mutation
bortezomib on the modulation of NF-jb activity by pp65 immunofluo-
(exon 4), and c-KIT mutation (exon 17) as previously described.51
rescence staining in patients with low or intermediate-1 MDS. The secondary objectives were to determine the clinical activity, safety and
2.5 | Study design and treatment plan
tolerability of SC bortezomib in patients with low or intermediate-1 MDS. The primary efficacy outcome was the overall response (OR) rate
This was a single arm proof of concept study designed primarily to determine the effect of SC bortezomib on modulation of NF-jb activity, and secondarily to determine the clinical activity and safety and tolerability of SC bortezomib in patients with low or intermediate-1 risk MDS who had evidence of NF-jB activation. This study was approved by the University of Texas MD Anderson Cancer Center’s Institutional Review Board in accordance with an assurance filed with and approved by the Department of Health and Human Services. Informed written consent was obtained from each patient before enrollment in the study. This trial is registered at www. clinicaltrials.gov as NCT01891968. Bortezomib was administered via SC route at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11 of each cycle. The duration of each treatment cycle was 21 days. Patients received the first cycle of therapy without interruption regardless of the degree of myelosuppression. After the first course of therapy, the interval between cycles of therapy could be spaced out at the discretion of the treating physician. The dose and schedule were adapted based on PK and safety data for SC bortezomib as published by Moreau et al.37 Doses were held and sequentially reduced for treatment-related adverse events, as defined in the protocol. The first dose reduction level was 1 mg/m2 and the second dose
based mainly on hematologic improvement as defined by the IWG, but could include complete remission, partial remission and marrow complete remission. A maximum of 40 evaluable patients could be enrolled, but the study would be subject to premature discontinuation if the data suggested that: Pr (h < 0.15|data)>0.95 where h was the OR rate. Currently the standard practice in low risk MDS is observation; therefore, an OR rate of about 15% was worth considering. We assumed the OR rate had a prior Beta distribution (0.3, 1.7) with mean of 0.15 and variance of 0.0425. The study was to be discontinued prematurely if at any time we determined that there was a >95% chance that the average OR rate was
