Leukemia Research 27 (2003) 375–380
`
`Pilot study of recombinant human soluble tumor necrosis factor
`(TNF) receptor (p75) fusion protein (TNFR:Fc; Enbrel) in
`patients with refractory multiple myeloma: increase
`in plasma TNF␣ levels during treatment
`Apostolia-Maria Tsimberidou a, Tracey Waddelow a, Hagop M. Kantarjian a,
`Maher Albitar b, Francis J. Giles a,∗
`
`a Department of Leukemia, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
`b Department of Hematopathology, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
`
`Received 28 January 2002; accepted 23 April 2002
`
`Abstract
`Elevated tumor necrosis factor (TNF)-␣ levels are associated with poor prognosis in patients with multiple myeloma (MM). Enbrel
`is a TNF antagonist fusion protein consisting of the extracellular, ligand-binding domain of the human p75 TNF receptor linked to the
`Fc portion of human IgG1. Ten patients with refractory MM were treated with Enbrel 25 mg s.c twice weekly for a minimum of eight
`median age was 63 years (range, 43–76). The total number of Enbrel doses was 191 (median 16; range, 3–55). TNF␣ plasma levels
`increased significantly during treatment with Enbrel. No objective response occurred. Acceleration of disease occurred in four patients.
`While well-tolerated, Enbrel did not have anti-myeloma activity as administered on this study.
`© 2002 Elsevier Science Ltd. All rights reserved.
`
`Keywords: Refractory; Multiple myeloma; TNF␣; Enbrel
`
`1. Introduction
`
`The management of patients with relapsed or refractory
`multiple myeloma (MM) remains inadequate and novel
`treatment modalities are urgently needed. The response rate
`with standard therapy, including combination chemotherapy
`with vincristine, adriamycin, and dexamethasone (VAD)
`is approximately 60% [1–3]. Thalidomide single-agent
`therapy is associated with overall response rates of approx-
`imately 30% and 2 years overall and failure-free survival
`rates of 48 and 20%, respectively [4,5].
`Among the potential MM growth factors, tumor necro-
`sis factor (TNF)-␣ is a survival factor for MM cell lines,
`induces MM cells in the cell-cycle and promotes long-term
`growth of malignant plasma cells [6]. It promotes the
`growth of MM cell lines, sometimes in a synergistic manner
`with interleukin-6 (IL-6), but also may clearly act through
`a pathway independent of IL-6, having a growth-promoting
`effect at least equal to that of IL-6 [7–11]. TNF␣ is also a
`potent bone-resorbing factor and plays an important role in
`
`∗
`
`Corresponding author. Tel.: +1-713-792-8217; fax: +1-713-794-4297.
`E-mail address: fgiles@mdanderson.org (F.J. Giles).
`
`the development of the osteolytic bone lesions observed in
`MM patients [12–15]. In some models, the role of TNF␣ in
`MM is more complex; it stimulates both growth and apopto-
`sis of some plasma cell lines and some ex-vivo plasma cells
`[8,16]. Fillela et al. found that TNF␣ serum levels were in-
`creased in 44% of patients with newly diagnosed MM and
`50% of those with progressive disease [11]. TNF␣ serum
`levels were significantly higher in persons with monoclonal
`gammopathy of undetermined significance (MGUS), or pa-
`tients with progressive MM compared with healthy subjects;
`patients with progressive MM also had significantly higher
`TNF levels than patients with stable MM. Furthermore,
`concentrations of TNF␣ are significantly higher in patients
`with bone disease than in those without overt lesions [17].
`Two distinct receptors for TNF of 55 and 75 kDa have
`been identified [18,19]. A recombinant TNF receptor p75-Fc
`fusion protein (Enbrel, Immunex, Seattle) was developed tar-
`geting to neutralize TNF, reducing its biologic activity [20].
`DNA encoding the Fc portion of a human immunoglobulin
`(Ig) G1 molecule was linked to DNA encoding the soluble
`portion of human p75 TNF receptor. The combined DNA
`was expressed in a mammalian cell line, resulting to an
`Ig-like dimer. This soluble TNFR-Fc fusion construct acts
`
`0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
`PII: S 0 1 4 5 - 2 1 2 6 ( 0 2 ) 0 0 0 8 2 - 6
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`
`as a competitive inhibitor of TNF, preventing its binding to
`the cell surface TNF receptors; it also renders it biologically
`unavailable [20].
`Studies in healthy normal volunteers and in patients with
`rheumatoid arthritis [21–24] Wegener’s granulomatosis [25]
`and advanced heart failure have shown that Enbrel is safe
`[21–28]. However, in patients with established septic shock
`caused by Gram-positive organisms there was a non-signifi-
`cant trend toward increased rates of mortality in those treated
`with higher doses of Enbrel in comparison with the placebo
`group; a similar tendency to increased mortality rates was
`also noted with the use of an anti-TNF monoclonal antibody
`on a prior study in a similar patient population [29–31].
`An effective anti-TNF agent might be of therapeutic
`benefit in patients with MM. As an initial investigation of
`the safety of Enbrel in this immunocompromised popula-
`tion, already prone to sepsis, we conducted a pilot study
`of Enbrel, as a single agent, in patients with advanced or
`refractory MM. Measurements of TNF␣, vascular endothe-
`lial growth factor (VEGF), basic fibroblast growth factor
`(bFGF), hepatocyte growth factor (HGF), and IL-6 were
`performed before and during treatment with Enbrel.
`
`2. Materials and methods
`
`2.1. Study group
`
`Patients with refractory MM were entered onto the study
`between August and December 2000, after written informed
`consent was obtained according to institutional guidelines.
`Refractory MM was defined as: (a) primary resistant MM,
`progressive disease during receipt of at least two courses
`of induction chemotherapy, which includes an alkylating
`agent and/or a topoisomerase II inhibitor; (b) transient
`response, defined as response but relapse while still on in-
`duction therapy; or (c) relapsed disease, i.e. post-remission
`or -plateau relapse Eligibility criteria included patients with
`a quantifiable serum paraprotein or Bence-Jones protein-
`urea and a bone marrow plasmacytosis >5%, without overt
`infection, hypotension, concurrent chemotherapy, systemic
`radiotherapy, pregnancy or overt psychosis.
`Pretreatment evaluation included history taking and phys-
`ical examination; complete blood count, differential, and
`platelets count; serum chemistries, including liver and renal
`function studies; bone marrow aspiration with or without
`biopsy; ␤2-M, serum immunoelectrophoresis, serum pro-
`tein, immunoglobulin assay and M-band quantitation by
`immunofixation, 24 h urine collection for Bence-Jones pro-
`tein, total protein, and creatinine; and radiologic assessment
`as indicated.
`
`2.2. Measurement of cytokine levels
`
`2.2.1. Plasma and serum collection
`Plasma and serum samples were collected and stored ac-
`cording to approved protocols from eight patients on study
`
`who consented to provide these specimens for cytokine as-
`say prior to first Enbrel therapy, at 2–3 week intervals while
`on study, and after completion of study therapy.
`
`2.2.2. Enzyme-linked immunosorbent assay
`The enzyme-linked immunosorbent assays (ELISAs) for
`TNF␣, VEGF, bFGF, HGF, and IL-6 were performed using
`commercially available kits from R&D Systems (Minneapo-
`lis, MN). Manufacturer’s recommended protocols were fol-
`lowed. Briefly, plasma was collected in tubes with EDTA
`and stored at −82
`◦
`C. Patient samples were added to separate
`microplates, each containing a specific monoclonal antibody
`and mixtures were incubated at room temperature for 2 h.
`The plates were washed three times to remove any unbound
`substances. Protein-specific enzyme-linked polyclonal anti-
`bodies were added to the wells. Subsequently, the mixtures
`were incubated at room temperature for 2 h followed by an-
`other washing to remove any unbound antibody or enzyme
`reagent. A substrate solution was added to the wells, and
`a blue color developed. The intensity of the blue was pro-
`portionate to the amount of cytokine bound in the initial
`step. The color development was stopped, and the intensity
`of the color was measured and compared with a standard
`curve. Reading was done at 450 nm wavelength for TNF␣,
`VEGF, bFGF, HGF, and IL-6.
`
`2.3. Therapy
`
`Treatment consisted of Enbrel 25 mg twice weekly sub-
`cutaneously (s.c.) for a minimum of eight doses (4 weeks;
`one cycle). If patient developed toxicity of grade 3–4 (NCI
`toxicity criteria), Enbrel was held until resolution to at least
`grade 1. Supportive care, including transfusion of blood and
`blood products, antibiotics, and analgesics were adminis-
`tered as needed.
`
`2.3.1. Course timing
`Enbrel was given for one course of treatment (4 weeks);
`if patients responded or had no signs of progression they
`received 16 additional doses (two courses) of Enbrel without
`interruption, at the same dose. Further courses were given
`if patients continued to respond or not to progress.
`
`2.4. Endpoints and statistical methods
`
`Complete response (CR) was defined as disappearance
`of serum and urine M-protein on electrophoresis and im-
`munofixation in two determinations at least 4 weeks apart,
`<5% plasma cells in the bone marrow, normalization
`of peripheral blood values or biochemical abnormalities
`assignable to MM, and resolution of all soft tissue plasma-
`tocytomas.
`Partial response (PR) was defined as ≥50% reduction of
`serum M-protein; ≥50% reduction in the urine M-protein
`if the baseline value was ≥1 g/24 h and <0.1 g/24 h if base-
`line value was 0.5–1 g/24 h; and ≥50% reduction of sum
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`
`of the products of the cross diameters of each measurable
`lesion. Disease progression was defined as ≥50% increase
`in the serum or urine M-protein above the lowest previous
`level, and appearance of new plasmatocytomas or increase
`by ≥50% of soft tissue plasmatocytomas. Failure to meet
`criteria for response or progression was categorized as sta-
`ble disease.
`Toxicity was graded on a scale of 0–5 using the National
`Cancer Institute Common Toxicity Criteria (NCI-CTC) Ver-
`sion 2.0 criteria [32].
`
`3. Results
`
`3.1. Study group
`
`The clinical characteristics of the 10 patients are summa-
`rized in Table 1. Median age was 63 years (range, 43–76;
`70%) were older than 60 years. Eight patients (80%) were
`male; one had a performance status (PS) of 2 (10%). Five
`patients had progressive and five stable/refractory MM.
`
`Table 1
`Patients’ characteristics
`
`Characteristic
`
`Age
`Median
`Range
`>60
`
`Male
`PS >1
`High ␤2M (>3 mg/l)
`
`Immunoglobulin type
`IgG
`IgA
`IgG Kappa chain deposition disease
`
`Marrow involvement
`
`Prior regimens
`0–2
`3–7
`
`Hb <10 g/dl
`WBC <1 × 109 l
`−1
`PLT <100 ×109 l
`−1
`M-protein >3 g/dl
`
`Bone lesions
`0–2
`>3
`
`Karyotype
`Diploid
`48–49, XY, −1,
`+add(3)(p26), −11,
`del(13)(q12q14),
`+14, −17 × 2,
`+19, −22, +4mar
`Prior thalidomide
`Prior hyper-CVAD
`Prior allogeneic transplant
`
`N = 10
`
`63
`43–76
`7
`
`8
`1
`8
`
`7
`3
`1
`
`6
`
`1
`9
`
`4
`3
`2
`5
`
`3
`7
`
`4/5
`1/5
`
`10
`6
`2
`
`%
`
`70
`
`80
`10
`80
`
`70
`30
`10
`
`60
`
`10
`90
`
`40
`30
`20
`50
`
`30
`70
`
`80
`20
`
`100
`60
`20
`
`Median number of prior treatments was five (range, 2–7).
`All patients had received prior therapy with thalidomide, six
`had received fractionated cyclophosphamide, vincristine,
`doxorubicin, and dexamethasone (Hyper-CVAD) regimen,
`five had received high-dose melphalan, two had received
`stem cell transplant; two had received Biaxin, and two pa-
`tients had received IFN-␣. The maximum response to prior
`treatment was CR in one patient, PR in six patients, and SD
`in three patients. One patient had IgG Kappa chain deposi-
`tion disease and Guillen-Barre like syndrome, six had IgG
`and three patients had IgA MM. Five patients had Stage I,
`two Stage II, and three Stage III disease as per the Durie
`and Salmon classification [33]. All patients with Stage I
`disease had received prior therapy for their disease based
`on symptoms attributable to their disease, usually fatigue
`and/or bone pain. Prior medical history was significant
`for recurrent severe infections in three patients: one had
`sinusitis, one urinary track infection due to ␤-hemolytic
`streptococcus, and one patient had bronchitis. The median
`Hgb value was 11 g/dl (range, 8.9–13.8); the median WBC
`4.7× 109 l
`−1 (range, 2.9–5.6) and the median platelet count
`was 199 × 109 l
`−1 (range, 57–356). The median M-protein
`was 2.7 g/dl (range, 0–4.7; M-protein was zero in a patient
`with Bence-Jones proteinuria, bone marrow infiltration, and
`>3 bone lytic lesions). Six patients had bone marrow infil-
`tration. Two patients had 0–1 bone lesions, one patient 2,
`and seven patients had >3 bone lesions. The median cre-
`atinine was 1.0 mg/dl (range, 0.7–2.6), the median ␤2-M
`6.9 mg/l (range, 1.2–17.5) and the median serum calcium
`was 8.7 mg/dl (range, 7.7–10.5). Cytogenetic studies were
`successful
`in five patients; four had diploid karyotype
`and one patient had multiple chromosome abnormalities
`(48–49, XY, −1, +add (3)(p26), −11, del (13)(q12q14),
`+14, −17 × 2, +19, −2, +4mar).
`
`3.2. Treatment results
`
`Ten patients received a total of 25 cycles of Enbrel therapy.
`The median number of cycles administered was 2 (range,
`1–7). The median number of doses was 16 (range, 3–55)
`and the total number of doses was 191.
`
`3.2.1. Response
`No patient had a complete or partial response to therapy.
`Four patients had progressive disease on study, including
`two patients who were withdrawn early (after 2 and 4 weeks;
`Table 2). Among the four patients who progressed, three
`patients had stable MM on study entry.
`
`3.3. Cytokine levels
`
`Cytokine plasma levels were measured in eight patients
`who agreed to provide samples before and during treatment
`with Enbrel (Table 3). TNF␣ plasma levels were signifi-
`cantly higher during Enbrel treatment compared with the
`levels before treatment. In contrast, there was no significant
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`Table 2
`Response
`
`A.-M. Tsimberidou et al. / Leukemia Research 27 (2003) 375–380
`
`Patients
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`MM status on
`Duration of
`M-protein
`entry
`Rx (weeks)
`baseline
`SD/refractory
`12
`0.10
`PD
`8
`3.70
`PD
`26
`3.20
`SD/refractory
`4
`1.30
`PD
`2
`0.13
`SD/refractory
`9
`4.70
`SD/refractory
`12
`4.00
`PD
`3
`2.20
`PD
`8
`0.30
`SD/refractory
`27
`3.40
`a Improvement in Hgb from 9.7 to 10.4 g/dl, WBC (4.4–5.8) × 106 l
`b Progressive growth of myeloma slowed.
`c Early removal from the study.
`
`M-protein min
`M-protein
`M-protein
`during Rx
`(end of Rx)
`increase (%)
`0.20
`0.20
`50
`4.30
`4.80
`30
`3.50
`3.50
`9
`2.20
`2.20
`69
`0.42
`0.56
`460
`4.50
`5.30
`13
`4.00
`6.80
`70
`2.20
`3.10
`41
`0.30
`0.30
`0
`3.20
`3.50
`3
`−1, bone marrow plasma cells reduced from 32 to 18%.
`
`Response
`
`PD
`SDa
`SDb
`PD
`PDc
`SD
`PD
`SD
`SD
`SD
`
`Table 3
`Cytokine levels in plasma of patients treated with Enbrel
`Mean (±2S.D.)
`
`Cytokine levels
`
`Median (range)
`
`TNF␣
`Pretreatment
`During treatment
`
`VEGF
`Pretreatment
`During treatment
`
`Bfgf
`Pretreatment
`During treatment
`
`HGF
`Pretreatment
`During treatment
`
`IL-6
`Pretreatment
`During treatment
`
`8.0 (6.7–9.1)
`243.6 (72.7–413.9)
`
`77.7 (38.5–294.1)
`68.3 (43.4–91.4)
`
`20.4 (8.6–57.8)
`17.2 (7.3–25.2)
`
`7.9 (±1.4)
`250.8 (±243.93)
`
`118.3 (±196.7)
`64.6 (±39.4)
`
`25.4 (±38.0)
`16.0 (±16.3)
`
`786.3 (540.3–1527.5)
`743.2 (371.8–2009.3)
`
`855.1 (±743.6)
`898.3 (±1148.4)
`
`2.7 (2.4–8.9)
`3.4 (1.9–8.4)
`
`3.89 (±7.7)
`4.08 (±5.6)
`
`P-value
`
`0.006
`
`0.20
`
`0.22
`
`0.63
`
`0.44
`
`difference before and during treatment with Enbrel in the
`plasma levels of VEGF, bFGF, HGF, and IL-6.
`
`3.4. Toxicity
`
`Enbrel was associated with grade 2 fever in two patients;
`grade 1 fatigue in one; grade 2 flu-like syndrome in two;
`grade 2 chest pain in one; grade 2 abdominal discomfort in
`one; and grade 1 hyperbilirubinemia in one. There were no
`overt allergic reactions to Enbrel. No patient was withdrawn
`from the study because of toxicity. There was no increased
`mortality rate in patients treated with Enbrel. No patients
`developed sepsis while on study.
`
`4. Discussion and conclusion
`
`The administration of Enbrel at the dose of 25 mg s.c.
`twice weekly was not associated with overt serious adverse
`
`events in these patients with heavily pretreated refractory
`MM. There was no evidence of cumulative toxicity, and the
`more common adverse events were fever and flu-like syn-
`drome. More importantly, there was no increased mortal-
`ity rate among patients treated with Enbrel. However, no
`responses were observed. Four patients progressed and six
`patients had stable disease.
`The safety profile of Enbrel in patients with MM in our
`study is in line with other reports in patients with rheuma-
`toid arthritis [21–24], Wegener’s granulomatosis [25], and
`advanced heart failure, [27,28] showing that Enbrel
`is
`well-tolerated. The most common side effects, such as in-
`jection site reactions and upper respiratory tract infections,
`seen in other disorders, were not noted in our study popula-
`tion [34,35]. The stimulus to perform the currently reported
`pilot safety study was the observation of a trend toward
`increased mortality rates with higher doses of Enbrel com-
`pared with a placebo group in patients with documented
`sepsis from Gram-positive bacteria [29]. The same trend has
`been observed in two trials of an anti-TNF␣ monoclonal
`antibody for the treatment of sepsis: in non-shock patients
`receiving a 15 mg/kg dose in one study [30], and in shock
`patients treated with the same dose in a second study [31].
`Enbrel is a dimer of the p80 TNF receptor linked by the Fc
`portion of IgG1, which binds TNF␣ and lymphotoxin, neu-
`tralizing their effects. This dimeric construct of Enbrel has a
`higher affinity for TNF than the monomeric forms of the re-
`ceptor. Additionally, the Fc peptide gives a longer half-life to
`the molecule [20]. The primary mechanism of its action is by
`binding to the TNF␣, rendering it biologically unavailable.
`Preclinical and clinical studies have shown that Enbrel does
`not cause rapid removal of TNF from the biologic fluids, but
`does prolong TNF’s half-life [20,36]. Enbrel has been re-
`ported to act as a TNF “carrier” [21,36]. This “carrier” activ-
`ity of Enbrel may explain the finding of significantly higher
`TNF␣ values in patients during treatment on this study
`compared with their respective pretreatment values. This
`observation is in agreement with the study of Eason et al.,
`who showed similar effects in patients with OKT3-acute
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`clinical syndrome [36]. These investigators demonstrated
`that the high TNF␣ antigenic levels were associated with
`concomitant low or undetectable TNF␣ bioactivity; high
`levels of TNF receptors were also noted >13 days after the
`administration of Enbrel, indicating its long half-life [36].
`Despite this data suggesting that the elevated TNF␣ levels
`associated with Enbrel use are not bioactive, some caution
`must be applied in accepting that this is always so. A note-
`worthy event on the current study was the acceleration of
`MM in four patients soon after commencing Enbrel therapy,
`three of whom had entered on study with an immediate prior
`history of stable disease. In this study, we focused on safety
`in terms of lack of overt adverse events—Enbrel was clearly
`“safe” from this perspective. However, its safety in terms
`of modulation of disease activity in patients with MM will
`require much more attention in other studies. The source
`of the elevated circulating TNF␣ in patients with MM re-
`ceiving Enbrel is of interest. Serial quantitative RT-PCR
`analyses of mRNA expression for relevant cytokines in both
`myeloma and stromal cells would be of benefit in future
`studies.
`Neben et al. have investigated the genetic polymorphism
`in the TNF␣ in patients with relapsed and refractory MM
`treated with thalidomide [37]. Eight patients with MM car-
`rying the −238A allele had higher TNF␣ levels in peripheral
`blood, prolonged 12 months progression free survival and
`a trend towards longer overall survival compared with pa-
`tients with the −238G allele [37]. Among patients with the
`−238G allele, only one patient had achieved a CR. These
`investigators suggest that regulatory polymorphisms of the
`TNF␣ gene can affect TNF␣ production and the response to
`thalidomide. Of particular interest is the fact that all patients
`in our study had previously failed thalidomide; although no
`studies for genetic polymorphism were performed, it is pos-
`sible that patients who progressed may have been carriers
`of the −238G allele.
`Enbrel is also being investigated in patients with other
`hematologic malignancies. In a cohort of seven patients with
`acute myelogenous leukemia (AML), a single s.c. 25 mg
`dose resulted to a reduction of apoptosis in three out of five
`evaluable patients and increase of proliferation in three out
`of five patients [38]. The drug was well-tolerated without
`any side effects. In six patients, the WBC count stabilized
`or decreased, but no patient achieved an objective response.
`In patients with myelodysplastic syndromes, the combi-
`nation of Enbrel with thalidomide was well-tolerated, and
`produced significant hematologic improvement in 4 out of
`18 patients who completed 16 weeks of therapy [39]. Five
`patients had stable disease and three had a major erythroid
`response. In a pilot study in patients with myelofibrosis with
`myeloid metaplasia, Enbrel relieved constitutional symp-
`toms and was well-tolerated but no objective responses
`were documented [40]. In a Phase 2 study of Enbrel, in 26
`patients with refractory myeloproliferative malignancies,
`the agent was very well-tolerated, but no patients had a
`clinically meaningful response to therapy [41].
`
`In conclusion, Enbrel had an acceptable safety profile
`in patients with refractory MM. As a single agent it did
`not
`induce any remissions. This pilot study involved a
`small patient cohort and thus, its findings are not definitive.
`Longer-term follow-up of a larger patient cohort would be
`required to properly assess any relationship between the
`increased levels of plasma TNF␣ associated with Enbrel
`therapy and disease behavior in patients with MM.
`
`Acknowledgements
`
`All authors analyzed and interpreted the data and gave
`final approval of the article. In addition, A.-M. Tsimberi-
`dou helped draft the article, provided critical revision of the
`article for important intellectual content, and collected and
`assembled the data. T. Waddelow provided administrative,
`technical or logistical support and collected and assembled
`the data. H.M. Kantarjian contributed to the conception
`and design and critical revision of the article in addition to
`providing statistical expertise and administrative, technical,
`or logistic support. M. Albitar also contributed to critical
`revision of the article for important intellectual content,
`provided study materials or patients, and offered adminis-
`trative, technical, or logistic support. F.J. Giles contributed
`to the conception and design, helped draft the article, as-
`sisted with critical revision, provided study materials or
`patients, and obtained necessary funding.
`
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