Haematologica 2003 - Supplements

P4.5
CYTOKINES IN MGUS: THERAPEUTIC
INTERVENTIONS TO PREVENT PROGRESSION
John A. Lust, M.D., Ph.D. Kathleen A. Donovan, Ph.D.
Mayo Clinic, Rochester, MN
In previously published work, we demonstrated that detection of IL-
1β expression by RT/PCR or ISH was useful at differentiating active
MM from MGUS. However, we were unable to differentiate the
clinically benign condition, SMM from active MM using RNA based
techniques. Therefore, we hypothesized that quantitative differences
in IL-1β function may differentiate SMM from active MM.
Measurement of IL-1β concentration by ELISA proved to be
inadequate because of insufficient sensitivity. More importantly,
ELISA measured only IL-1β concentration whereas IL-1β functional
activity is modulated by several IL-1 family members such as IL-1
receptor antagonist and soluble IL-1 receptor. Therefore, we
measured IL-1β functional activity with a bioassay using IL-6
production by bone marrow stromal cells as a highly sensitive
surrogate marker. IL-1β bioactivity was determined by quantitating
IL-6 production by cultured bone marrow stromal cells using an IL-6
ELISA. Ficoll purified bone marrow cells from patients with various
plasmaproliferative disorders were cultured at 2 x 10 6 cells/ml for 48
hours. Supernatants were pooled, aliquoted and frozen at –80 0 C.
Normal stromal cells (Clonetics, Walkersville, MD) were plated at 1
x 10 5 cells/ml and incubated at 37 0 C for 48 hours. After 48 hours, the
stromal cells were washed and either IL-1β standards or patients’
supernatants were added with or without an IL-1β inhibitor (anti-IL-
1β antibody or IL-1 receptor antagonist). Cultures were incubated at
37 0 C for another 48 hours. Supernatants are harvested and frozen at -
80 0 C for analysis and subsequently analyzed for IL-6 using the
Biosource ELISA kits according to the manufacturer’s specifications.
Response of the stromal cells to IL-1β was calibrated with a standard
curve using recombinant IL-1β. These cells respond to IL-1β in a
sigmoid fashion with as little as 1 pg/ml of IL-1β inducing 20,000
pg/ml of IL-6.
Culture supernatants of unsorted bone marrow cells from 77
untreated patients (2 normal, 12 MGUS, 18 MM, 45 SMM/IMM)
were tested in this assay. Results were expressed as fold increase
= [patient IL-6 – media control IL-6]/media control IL-6. A fold
increase was utilized to allow for interassay comparison between
different batches of stromal cells. IL-1β specificity was
determined by inhibition of the IL-6 production with anti-IL-1β
antibody. IL-1 specific IL-6 production showed that myeloma
patients induced a 2.2 - 24.1 fold increase in IL-6 that was
statistically different from the -0.25 - 1.9 fold increase by
normal/MGUS patients (p90% in
41/53 patients tested (100% in 32/53). A logistic regression
analysis was performed on 62 of the 75 patients that had been
followed for at least a year or progressed during that time.
Variables examined were % bone marrow plasma cells, plasma
cell labeling index, albumin, M-protein level, beta-2
microglobulin, creatinine, calcium, hemoglobin, and stromal cell
IL-6 production. Only two factors, stromal cell IL-6 production
and bone marrow plasma cells, were found to be significant (p <
.01) in predicting progression of SMM to myeloma.
Subsequently, a Kaplan-Meier estimate was performed on the 45
patients with SMM/IMM. The time to progression was defined
as the time from the date of the bone marrow on which the IL-1β
bioassay was performed to the date of progression to active MM.
Seventeen patients with SMM/IMM had a fold increase less than
1.6 and, to date, none of these patients have progressed to active
disease. In contrast, 28 patients with SMM/IMM demonstrated a
FI > 1.6 and 14 of these 28 patients have progressed to active
MM with a median time to progression of approximately 2.5
years.
Bone marrow cells from a patient with MM were sorted into CD138+
plasma cells and CD138- populations and examined for IL-6
production by the IL-1β stromal cell bioassay. The IL-6 production
was induced predominantly by the CD138+ plasma cells. These
results correlate with our previously published data by ISH
demonstrating that the monoclonal plasma cells from virtually all
myeloma patients express IL-1β mRNA. In addition, the IL-6
production was significantly inhibited with IL-1 receptor antagonist.
The above studies served as the preclinical data for a Phase II trial of
IL-1 receptor antagonist (IL-1Ra) in patients with SMM/IMM.
We have now accrued eight patients in the clinical study using
IL-1Ra in patients with SMM/IMM and preliminary studies are
available on four. Patients that have > 10% bone marrow clonal
plasma cells and/or an M-spike > 3 g/dL and do not require
immediate chemotherapy are eligible. Patients receive 100 mg of
Anakinra SQ qd. Clinical results are available on four patients
with 1-3 months of follow-up. The underlying hypothesis is that
IL-1β stimulates paracrine IL-6, the major growth factor for
myeloma cells and that IL-1 receptor antagonist (IL-1Ra) will
inhibit paracrine IL-6 production ultimately leading to apoptosis
of the myeloma cell. Using CRP as a surrogate marker for IL-6
levels, all four patients demonstrated a reduction in CRP levels
For example, Patient 1 had a reduction in CRP (mg/dL) from 3.82
to 0.825 to 0.122 or a 96% reduction; patient 2 a 74% reduction
in CRP after 2 months. In a similar fashion, urine NTX
(nmole/L), which is a marker of osteoclast activity was decreased
in three patients (a followup urine was not available on one of the
patients). Patient #1 has also had an increase in her hemoglobin
from 10.1 to 11.9. Monoclonal protein levels have remained
stable in three of the patients so far. However, patient #2
demonstrated a gradual decline in the CRP with IL-1Ra that
paralleled a reduction in the serum monoclonal protein (see
Figure). Toxicity has been minimal with mild injection site
reactions during the first month of therapy. One patient developed
an asymptomatic Grade 4 neutropenia and the IL-1Ra was held.
The patient’s counts returned to normal within 1 week and the IL-
1Ra was restarted at 100mg qod. Although preliminary, these
results demonstrate that IL-1Ra has biologic activity in patients
with SMM/IMM and support the hypothesis that upregulation of
IL-1β production is a critical event in the transition of
SMM/IMM to active myeloma.
Donovan KA, Lacy MQ, Kline MP, Ahmann GJ, Heimbach JA, Kyle RA,
Lust JA:. Contrast in cytokine expression between patients with
monoclonal gammopathy of undetermined significance or multiple
myeloma. Leukemia 12:593-600, 1998
Lust JA, Donovan KA: Novel therapeutic strategies. Cancer Research
Therapy and Control 6:225-226, 1998.
Lacy MQ, Donovan KA, Heimbach JK, Ahmann GJ, Lust JA:
Comparison of interleukin-1β expression by in situ hybridization in
monoclonal gammopathy of undetermined significance and multiple
myeloma. Blood 93(1):300-305, January 1, 1999.
Lust JA and Donovan KA: Role of IL-1β in the Pathogenesis of Multiple
Myeloma. Hematology/Oncology Dec. 1999, 13(6):1117-1125.
Donovan KA, Lacy MQ, Gertz MA, Lust JA. IL-1β Expression in IgM
Monoclonal Gammopathy and Its Relationship to Multiple Myeloma.
Leukemia, March 2002, 16:382-385.
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