Multiple Myeloma: New Insights and Therapeutic Approaches
Kenneth C. Anderson (Chair), Robert A. Kyle, William S. Dalton,Terry Landowski, Kenneth Shain,
Richard Jove, Lori Hazlehurst, and James Berenson
This review discusses the evolution of novel
new bone formation in the marrow milieu have
diagnostic and treatment strategies for multiple
already derived effective bisphosphonate therapy.
myeloma based upon increased understanding of
These drugs not only reduce bone complications
basic disease pathogenesis. Although myeloma
and related pain, thereby improving quality of life,
has remained an incurable illness to date, these
but also may have intrinsic anti-tumor activity by
new developments will derive treatments to im-
virtue of inducing tumor cell adherence to marrow,
prove outcome and achieve eventual cure.
reducing interleukin-6 secretion, inducing tumor
In Section I, Dr. Kyle reviews the results of
cell apoptosis, or inhibiting angiogenesis.
current therapy for multiple myeloma, including
In the last section, Dr. Anderson explores the
high dose therapy and stem cell transplantation
potential for future therapies which offer great
which have proven to achieve improved response
promise to improve patient outcomes. First, drugs
rates, event-free, and overall survival. Supportive
which alter the marrow microenvironment include
therapy, such as erythropoietin to treat disease-
thalidomide and its derivative immunomodulatory
related anemia, and methods of prophylaxis against
drugs, which act directly on tumor cells to induce
infection, which both lessen toxicities of treatment
apoptosis or G1 growth arrest, alter tumor cell
and improve quality of life for patients, are also
adhesion to marrow stroma, inhibit angiogenesis,
addressed.
and trigger a cellular anti-tumor response. The
In Section II, Dr. Dalton with Drs. Landowski,
proteasome inhibitors both act directly on tumor
Shain, Jove and Hazlehurst discusses mechanisms
cells and also inhibit the transcription factor NF B-
of drug resistance in myeloma, with emphasis on
dependent upregulation of IL-6 secretion triggered
novel treatment approaches to prevent develop-
by tumor cell adhesion. Second, delineation of both
ment of drug resistance and to overcome drug
growth and apoptotic pathways has derived novel
resistance. Laboratory studies delineating mecha-
treatment strategies. Third, the preclinical basis and
nisms whereby myeloma cells resist drug-induced
early clinical trial results using vaccination and
apoptosis provide the framework for related treat-
adoptive immunotherapy to harness autoimmune
ment protocols for patients with refractory disease.
and alloimmune anti-myeloma responses are
In Section III, Dr. Berenson reviews the manage-
presented. This review sets the stage for an evolv-
ment of complications in bone, which occur in the
ing new biologically based treatment paradigm in
majority of patients with myeloma and are the
myeloma targeting both the tumor and its microen-
major cause of decreased quality of life. New
vironment to improve outcome and achieve even-
insights into the mediators of bone resorption and
tual cure.
I. CURRENT THERAPY OF MYELOMA
reevaluate the patient in two or three months and delay
Robert A. Kyle, M.D.*
therapy until progressive disease is evident.
If the patient is younger than 70 years, the physician
Although most patients with multiple myeloma (MM)
should discuss the possibility of an autologous periph-
have symptomatic disease at diagnosis and require
eral blood stem cell transplant with the patient. Hemato-
therapy, some are asymptomatic and should not be treated.
poietic stem cells should be collected before the patient
If there is doubt about beginning therapy, it is best to
is exposed to alkylating agents. Chemotherapy is the pre-
ferred initial treatment for symptomatic MM in persons
older than 70 years or in younger patients in whom trans-
* Mayo Clinic, Room 920 Hilton Bldg., 200 First Street SW, Roch-
ester MN 55905
plantation is not feasible.
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Peripheral blood stem cells are preferable to bone
and disease responsive to initial chemotherapy) but who
marrow transplantation because engraftment is more rapid
were treated with conventional chemotherapy had a sur-
and there is usually less contamination of the infused cells
vival of 5 years, which is similar to that seen in autolo-
with tumor cells. The absolute number of CD34+ cells is
gous stem cell transplantation.3
the most reliable and practical method for determining
In a series of 177 patients < 75 years of age with IgG
the efficacy of the stem cell product. Autologous periph-
myeloma, C-VAMP followed by high-dose chemotherapy
eral stem cell transplantation is applicable for more than
with or without stem cell rescue and maintenance inter-
half of patients with MM. The two major shortcomings
feron were reported. The median survival was 4.9 years.
are: 1) Eradication of myeloma from the patient rarely
Those with 2-microglobulin < 2.7 mg/L and those < 52
occurs even with large doses of chemotherapy and/or ra-
years of age had a more favorable response.4
diation and 2) autologous peripheral blood stem cells are
The role of TBI in the preparative regimen is contro-
contaminated by myeloma cells or their precursors. For-
versial. In a comparison of melphalan, 140 mg/m2 plus
tunately, mortality from autologous transplantation is only
TBI vs melphalan 200 mg/m2, there was no difference in
1­2% if patients are appropriately selected.
response rate, event-free survival and overall survival.
Most physicians initially treat the patient with vinc-
However, the toxicity of melphalan 200 mg/m2 was sig-
ristine and doxorubicin (Adriamycin) given IV for 96
nificantly lower than melphalan plus TBI.5 Consequently,
hours and dexamethasone orally (VAD) for 3­4 months
many have discontinued TBI and give only melphalan,
to reduce the number of tumor cells. Dexamethasone with
200 mg/m2 for the preparative regimen.
or without thalidomide is being evaluated for initial
The role of double or tandem autologous stem cell
therapy. Peripheral blood stem cells are collected follow-
transplants is controversial. In an uncontrolled series of
ing granulocyte colony-stimulating factor (G-CSF) with
231 newly diagnosed MM patients who received a sec-
or without high-dose cyclophosphamide. One can then
ond transplant, 51% achieved complete response and 95%
proceed with the transplant following high-dose chemo-
had a complete or partial response. The authors felt that
therapy and/or total body irradiation (TBI) followed by
the double transplant extended both event-free and over-
infusion of the peripheral blood stem cells. The other
all survival even in patients with unfavorable cytogenet-
choice is to treat the patient with alkylating agents after
ics and 2-microglobulin values.6 In a randomized trial
stem cell collection until a plateau is reached and then
of 400 patients from France, there was no difference in
give the patient alpha-2-interferon ( IFN) or no therapy
event-free or overall survival between single and double
2
until early relapse. At that time, the patient is given high-
autologous stem cell transplants when evaluated at two
dose melphalan with or without total body radiation and
years. The two groups were similar from the standpoint
the previously collected peripheral blood stem cells are
of age, gender, stage, Ig isotype, 2-microglobulin value,
infused. Early or late transplantation are reasonable op-
C-reactive protein level and bone marrow plasmacyto-
tions. In one study, 185 patients were treated with three
sis. The complete response rate was 32% with a single
or four courses of VAD and then randomized to high-
transplant and 33% with a double transplant. At 2 years,
dose chemotherapy and autologous stem cell transplan-
the event-free survival was 54% vs. 57% while the overall
tation or to conventional chemotherapy. There was no
survival was 71% vs 67%.7 In a subsequent evaluation, pa-
difference in median survival of the two groups (65 vs.
tients with a low 2-microglobulin value at diagnosis ap-
64 months). The main advantage of early transplantation
peared to have better results with the double transplant.
was a shorter period of chemotherapy.1
Almost all patients will relapse following an autolo-
A randomized trial by the French Myeloma Group
gous stem cell transplant. A preliminary analysis of a ran-
compared high-dose chemotherapy followed by autolo-
domized trial of 85 patients with MM who were treated
gous bone marrow transplantation with conventional che-
with high-dose melphalan and autologous bone marrow
motherapy in 200 previously untreated myeloma patients
transplantation followed by
-IFN maintenance therapy
2
under the age of 65 years.2 Data was analyzed on an in-
suggested both a relapse-free survival and overall sur-
tention-to-treat basis. Twenty-five percent of the patients
vival benefit. However, the final analysis of this trial dem-
randomized to transplantation did not receive a transplant.
onstrated no significant difference in relapse-free or over-
Response rate (81% vs 57%) and complete response rate
all survival among patients randomized to maintenance
(22% vs. 5%) were superior in the transplant group. The
therapy with
-IFN.8 Idiotype-treated autologous dendritic
2
5-year event-free survival (28% vs 10%) and overall sur-
cells are being used to prolong response duration.9
vival (52% vs 12%) were superior in the transplant group.
In an effort to prolong survival, highly purified
It must be kept in mind that patient selection plays an
CD34+ cells did not influence the achievement of clini-
important role in response and survival. In one report, 77
cal or molecular complete remission or remission dura-
patients with MM who fulfilled the criteria for transplant
tion or overall survival.10 Thus, tumor cell purging does
(age < 66 years, Stage II or III, good performance status
not appear to be beneficial.
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The use of
-IFN for more than six months follow-
conventional chemotherapy is controversial due to con-
2
ing autologous stem cell transplantation resulted in de-
flicting results and frequency of undesirable side effects.
lay of platelet recovery.11 Patients refractory to VAD had
In a large meta-analysis Wheatley reported a survival
the same overall survival as those who responded to
benefit in both induction (p = 0.05) and maintenance (p
VAD.12 In a retrospective study, high-dose therapy for
= 0.03) with an increase in median response duration of
newly diagnosed myeloma resulted in prolongation of
six months in both settings.18 Patients should be moni-
survival for patients < 60 years old when compared to
tored closely during the plateau phase and the same che-
historic controls treated with chemotherapy (61 vs 46
motherapy regimen should be reinstituted if relapse oc-
months).13
curs after six months. The use of prednisone, 50 mg orally
every 48 hours, appears to prolong the plateau state as
Allogeneic Bone Marrow Transplantation
well as the overall survival.19
The major advantage of allogeneic transplantation is that
the graft contains no tumor cells that can lead to a re-
Treatment of Refractory Multiple Myeloma
lapse. Unfortunately, over 90% of patients with multiple
Patients who are initially refractory or who become re-
myeloma are ineligible because of their age, lack of an
fractory to alkylating agent therapy have a modest re-
HLA-matched sibling donor or inadequate renal, pulmo-
sponse rate to subsequent chemotherapy and a limited
nary or cardiac function. Furthermore, there is a mortal-
survival. The highest response rates in patients with MM
ity of at least 25% at present.
resistant to alkylating agents have been with vincristine,
In a report of 266 patients from the European Blood
doxorubicin and dexamethasone (VAD). Vincristine and
and Bone Marrow Transplantation registry, 51% obtained
doxorubicin are given by continuous infusion for four
a complete response. The overall treatment mortality rate
days and dexamethasone (40 mg daily) is given on days
was approximately 40%. The actuarial survival was 30%
1­4, 9­12, and 17­20 each month. Dexamethasone is of-
at 4 years and 20% at 10 years.14
ten given only on days 1-4 in even-numbered cycles be-
It is obvious that the mortality rate for allogeneic
cause of toxicity. Dexamethasone can be given as the only
transplantation must be reduced before it can assume a
therapeutic agent since it accounts for approximately 80%
major role in the treatment of multiple myeloma. The
of the effect of VAD. Vincristine (0.4 mg) and doxorubi-
use of a "mini-allo" transplant15 or depletion of T cells in
cin (9 mg/m2) as a rapid intravenous infusion daily for 4
an effort to reduce transplant mortality are promising
days plus dexamethasone (40 mg) produced an objective
approaches. Graft-versus-myeloma effect has been noted
response in 67% of 134 previously untreated myeloma
after donor peripheral blood mononuclear cells were given
patients. This suggests that 4-day infusion of vincristine
for relapse following allogeneic transplantation. Eight of
and doxorubicin is unnecessary.20 Intravenous pulse me-
13 patients with relapsed myeloma following an alloge-
thylprednisolone (2 g three times weekly) is helpful for
neic bone marrow transplantation responded to donor
patients with pancytopenia and refractory disease. We find
lymphocyte infusions.16
fewer side effects from this approach than with dexam-
ethasone.21 Vincristine (2 mg), carmustine (BCNU; 30­
Chemotherapy
40 mg) and doxorubicin (30­40 mg) intravenously on
Various combinations of therapeutic agents have been
day 1 and prednisone daily for 5 days every 3 to 4 weeks
used because of the shortcomings of melphalan and pred-
produces benefit in about one-third of patients. Thalido-
nisone. Melphalan and prednisone produces an objective
mide produced responses in 32% of 84 patients with pre-
response in 50­60% of patients. In an overview of indi-
viously treated, progressive MM. After 12 months of fol-
vidual data from 4,930 persons from 20 randomized tri-
low-up, 22% of patients remained event free and 58%
als comparing melphalan and prednisone with a variety
were alive.22 Thalidomide was given in an initial dosage
of combinations of chemotherapeutic agents, the response
of 200 mg daily and gradually increased to 800 mg daily.
rates were higher with combination chemotherapy (60%)
Constipation, weakness or fatigue, sleepiness, skin rash
than melphalan and prednisone (53%) (p < 0.00001).
and peripheral neuropathy were undesirable side effects.
However, there was no significant difference in overall
In the majority of patients, response occurred within six
survival and there was no evidence that any group of pa-
weeks and with only 400 mg of thalidomide daily. The
tients benefited from receiving combination chemo-
use of thalidomide in conjunction with dexamethasone
therapy.17
is being explored.
Chemotherapy should be continued until the patient
is in a plateau state or for at least one year. Continued
chemotherapy may lead to the development of a myelo-
dysplastic syndrome or acute leukemia. The possible
benefit of maintenance therapy with
-IFN following
2
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Supportive Care
Hyperviscosity
This is characterized by oral or nasal bleeding, blurred
Renal failure
vision, paresthesias, headache or congestive heart fail-
Approximately 20% of patients with MM have a creati-
ure. It may occur from high concentrations of IgA or,
nine level > 2 mg/dL at diagnosis. The two major causes
rarely, IgG. The serum viscosity levels do not correlate
of renal insufficiency are "myeloma kidney" and hyper-
well with the symptoms or clinical findings. Conse-
calcemia. Dehydration, infection, nonsteroidal anti-in-
quently, a decision to perform plasmapheresis depends
flammatory agents and roentgenographic contrast media
on the symptoms and changes in the ocular fundus. Plas-
may contribute to acute renal failure. Amyloid deposi-
mapheresis promptly relieves the symptoms and should
tion occurs in 10% of patients who have MM and often
be done regardless of the viscosity level if the patient has
causes nephrotic syndrome, renal insufficiency or con-
signs or symptoms of hyperviscosity.28
gestive heart failure.
Maintenance of a high urine output (3 L/day) is im-
Emotional support
portant for preventing renal failure. Prompt treatment of
All patients with MM need substantial and continuing
hypercalcemia as well as correction of dehydration and
emotional support. The physician's approach must be
electrolyte imbalance is crucial. Alkalinization of the urine
positive in emphasizing the potential benefits of therapy.
may be useful.
It is reassuring for patients to know that some survive for
Acute renal failure should be treated with appropri-
10 years or more. It is vital that the physician caring for
ate fluid and electrolyte replacement and VAD in an ef-
patients with MM has the interest and capacity to deal
fort to reduce the tumor mass as quickly as possible. A
with an incurable disease over the period of years with
trial of plasmapheresis in younger patients with acute
assurance, sympathy and resourcefulness.
renal failure is a reasonable approach.23 Hemodialysis or
peritoneal dialysis is necessary in the event of symptom-
II. DETERMINANTS OF DRUG RESPONSE AND
atic azotemia.
DRUG RESISTANCE IN MULTIPLE MYELOMA
William S. Dalton, M.D., Ph.D.,* Terry Landowski,
Anemia
Kenneth Shain, Richard Jove and Lori Hazlehurst
Anemia occurs in almost all patients during the course of
MM. Fifty to sixty percent of patients respond to erythro-
Effective therapy for multiple myeloma began in the early
poietin.24,25 Improvement in the patient's quality of life and
1960s with the development of alkylating agents, in par-
an improved sense of well-being also occurs.26
ticular, melphalan and cyclophosphamide.1 Even today,
the combination of oral melphalan and prednisone is con-
Infection
sidered to be the mainstay of treatment for myeloma pro-
Patients should receive pneumococcal and influenza vac-
ducing responses in approximately 50­60% of patients.
cinations despite their suboptimal antibody response.
During the past four decades, various drugs have been
Prompt and appropriate therapy of bacterial infections is
found to be effective for the treatment of myeloma; most
essential. Prophylactic daily oral penicillin often benefits
of these drugs belong to the following pharmacologic
patients with recurrent pneumococcal infections. Since
classifications: alkylating agents (melphalan, cyclo-
many infections occur in the first two months after insti-
phosphamide, BCNU), topoisomerase II inhibitors (doxo-
tuting chemotherapy, the use of daily oral trimethoprim/
rubicin and etoposide), glucocorticoids (prednisone and
sulfamethoxazole is helpful.27 Intravenously administered
dexamethasone), and the anti-tubulin agent, vincristine.
gamma globulin may be beneficial for patients with re-
With the exception of the glucocorticoids, most of these
current infections, but it is inconvenient and very expen-
agents are relatively ineffective as single agents and are
sive.
best administered as a combination of agents. Since the
introduction of melphalan and prednisone, several drug
Spinal cord compression
combinations have been investigated. A popular combi-
This complication should be suspected in patients with
nation of drugs proven to be effective is the M2 protocol
severe back pain who develop weakness or paresthesias
consisting of vincristine, carmustine, melphalan, cyclo-
of the lower extremities or bladder or bowel dysfunc-
phosphamide and prednisone (VBMCP).2 While this com-
tion. Magnetic resonance imaging or computed tomog-
bination produces higher response rates than melphalan
raphy must be done immediately. An MRI is particularly
and prednisone, there is only a marginal improvement in
useful in demonstrating extramedullary plasmacytoma.
Radiation therapy and dexamethasone are usually effec-
tive, and surgical decompression is rarely necessary.
* Clinical Investigations, H. Lee Moffitt Cancer Center, 12902
Magnolia Drive, MRC 3043, Tampa FL 33612-9497
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overall survival that appears to occur chiefly for patients
Ras mutations. Mutations in the Ras family of genes are
with a poor prognosis.3 Similar observations have been
relatively common in myeloma patients. Neri and col-
made for other drug combinations including vincristine,
leagues first reported in 1989 that 32% of patients with
melphalan, cyclophosphamide, prednisone (VMCP) al-
myeloma had Ras mutations.7 The most frequent muta-
ternating with vincristine, carmustine (BCNU), doxoru-
tion occurred in the N-Ras gene, particularly at codon
bicin and prednisone.4 The combination of VAD (vinc-
61. The second most frequent mutation was found at
ristine, doxorubicin, and dexamethasone) produces re-
codon 12 for K-Ras. The frequency of Ras mutations in-
sponses in 60­70% of patients who become resistant to
creased from 27% at diagnosis to 46% with disease pro-
alkylating agents, but ultimately patients will develop
gression. Interestingly, these investigators found that pa-
resistance to all known chemotherapy regimens.5 The
tients with mutated Ras were less likely to respond to
problem of drug resistance is a major obstacle to curing
chemotherapy compared to patients with wild-type Ras.
myeloma and understanding factors that determine drug
In a more recent study of 346 newly diagnosed, untreated
response and the development of drug resistance should
patients, Liu et al reported a total incidence of 39% Ras
prove useful in developing means of preventing or over-
mutations.8 Patients with Ras mutations had a median
coming this problem.6
survival of 2.1 years compared to 4.0 years for patients
Substantial progress has been made in the last de-
with wild-type Ras. These studies demonstrate that Ras
cade in the identification of cellular mechanisms that
mutations adversely affect survival and may reduce re-
confer clinical drug resistance in myeloma. Research to
sponse to chemotherapy; therefore, targeting Ras signal-
date has chiefly focused on intrinsic cellular mechanisms
ing may be a novel therapeutic approach to the treatment
of drug resistance; in other words, mechanisms that re-
of myeloma.
side within the myeloma cell itself. Examples of intrin-
One approach to the interruption of Ras signaling in
sic cellular drug resistance include the following: (a) re-
the myeloma cell is to prevent Ras "processing," a re-
duction of intracellular drug concentration due to the
quirement for the activation of Ras.9 The enzyme
overexpression of membrane pump proteins such as P-
farnesyltransferase is responsible for transferring the
glycoprotein, (b) altered drug metabolism or enhanced
farnesyl group from farnesyl diphosphate, a cellular
drug detoxification, (c) alterations in the drug target that
chemical used in the synthesis of cholesterol, to certain
reduce drug efficacy, and (d) enhanced cellular repair of
proteins such as Ras. This process is important for Ras
drug-induced damage.6 Any one of these mechanisms
activation because it allows the protein to attach to the
could occur within a myeloma cell, and in fact, it is likely
inner plasma cell membrane. Preventing Ras from going
that a combination of these mechanisms occurs simulta-
to the plasma membrane by blocking its farnesylation
neously within the same myeloma cell. Studies both in
short-circuits oncogenic growth signals to the nucleus.
the laboratory and the clinic have demonstrated that elimi-
Inhibition of the farnesylation process is a prime target
nating or preventing a single mechanism of resistance,
for drugs aimed at preventing Ras activity.10 Recently,
such as reduced intracellular drug concentration due to
several drugs have been synthesized to inhibit protein
P-glycoprotein, selects for alternative mechanisms of drug
farnesylation. These drugs are called farneslytransferase
resistance. Based on this evidence, attempting to over-
inhibitors (FTIs) and preclinical studies have shown them
come a single mechanism of intrinsic drug resistance is
to be effective in tumors with Ras mutations. A phase II
not likely to produce long-standing clinical remissions.
clinical trial using the Janssen FTI (R11 5777) has been
In order to make substantial progress in the treatment of
approved by CTEP for the treatment of myeloma and
myeloma, it is important to develop a better understand-
should begin soon. A second approach to inhibiting Ras
ing of the biological principles that govern myeloma cell
processing may be the use of amino-bisphosphonates.
survival and growth. Understanding these principles will
Shipman et al found that the bisphosphonate incadronate
allow investigators to develop therapeutic approaches that
caused apoptosis in human myeloma cells by inhibiting
capitalize on the uniqueness of myeloma cells, thereby
the mevalonate pathway.11 This pathway is essential for
targeting the malignant cell and sparing normal cells.
the biosynthesis of sterols and long-chain isoprenoid lip-
ids including farnesyl PPi and geranylgeranyl PPi. These
Targeting Intrinsic Molecules and Pathways in the
latter two compounds serve as substrates for farnesyl-
Myeloma Cell
transferase and geranylgeranyl transferase, respectively,
One approach that may prove to be successful in improv-
and are transferred to small GTP-binding proteins such
ing myeloma therapy is the development of small mol-
as Ras. Inhibiting isoprenylation of these critical proteins
ecules that inhibit or interrupt cellular targets or path-
may induce apoptosis in myeloma cells.
ways that regulate myeloma cell growth and survival.
These pathways may be intrinsic to the myeloma cell it-
self, such as altered signal transduction pathways due to
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Targeting Extrinsic Molecules or Pathways Involved
inhibiting the production of IL-6, blocking the binding
in Myeloma Cell Survival and Growth
of IL-6 to its cell surface receptor, or by blocking down-
In addition to the intrinsic molecules or pathways dis-
stream signaling events within the myeloma cell itself.12
cussed above, there are signal transduction pathways that
One approach to blocking IL-6 receptor activation is to
rely on external communication between the myeloma
generate IL-6 variants that block the receptor in an inac-
cell and its environment. Similar to the intrinsic path-
tive form. One IL-6 variant in particular, called Sant7, is
ways, these extrinsic factors also regulate myeloma cell
a super-antagonist found to be effective at blocking IL-
survival and growth and are potential targets for novel
6.18 Preclinical testing of this super-antagonist is ongo-
therapy of myeloma. For example, the primary source of
ing in hopes that Sant7, or similar agents, may soon be
interleukin 6 (IL-6) is from the bone marrow stroma and
tested in the clinic (G. Ciliberto, personal communica-
not the myeloma cell itself.12 Inhibiting IL-6 production,
tion).
binding to its receptor, or downstream signaling may
Another approach to inhibiting IL-6 is to interrupt
block myeloma cell proliferation and result in apoptosis.
the intracellular signaling associated with cytokine re-
Myeloma cells express cell adhesion molecules that al-
ceptor activation. IL-6 induces intracellular signaling
low attachment and communication between the myeloma
through multiple pathways, but one pathway utilized by
cell and the bone marrow microenvironment.13 This com-
IL-6 to influence cell survival and resistance to apopto-
munication influences myeloma cell survival and growth;
sis is the JAK/STAT pathway. Engagement of cell sur-
interrupting cellular adhesion may induce programmed
face cytokine receptors activates the Janus kinase (JAK)
cell death and enhance the efficacy of standard treatment
family of tyrosine kinases, which in turn phosphorylate
of myeloma. In addition, it has recently been reported
and activate the cytoplasmic STAT (signal transducers
that myeloma cell growth and dissemination may depend
and activators of transcription) proteins.19 Activated
upon angiogenesis.14 Interactions between the microen-
STATs dimerize upon activation by JAKs and translo-
vironment and the myeloma cells may regulate angio-
cate to the nucleus where they bind specific DNA re-
genesis and stimulate myeloma growth and progression.
sponse elements and regulate the expression of certain
Blocking intercellular communications by blocking cell
genes. Seven mammalian STAT family members have
adhesion may represent a new approach for the treatment
been cloned and share common structural characteris-
of myeloma.
tics. One particular STAT member, Stat3, has been asso-
Figure 1 demonstrates two possible forms of inter-
ciated with oncogenesis when it is constitutively acti-
action between myeloma cells and the bone marrow mi-
vated.20 A recent report by Jove and colleagues demon-
croenvironment. A soluble form of interaction is repre-
strated that Stat3 is constitutively activated in bone mar-
sented by the interaction between IL-6, a cytokine pro-
row mononuclear cells in patients with myeloma.21 In
duced by the bone marrow stromal cells, and the my-
addition, Jove and co-workers demonstrated high levels
eloma cell. Interaction between the myeloma cell and the
of activated Stat3 in the myeloma cell line U266 known
bone marrow stroma mediated by cell adhesion molecules
to produce and utilize IL-6 for survival. Moreover, this
represents a direct form of interaction. For example, the
cell line is resistant to Fas-mediated apoptosis in spite of
interaction between cell surface integrins and extracellu-
high levels of Fas receptor being expressed in U266 cells.
lar matrix components, such as fibronectin, may be in-
Using specific inhibitors of the IL-6 receptor (Sant7), a
volved in prolonging cell survival and dissemination of
Jak2 inhibitor (AG490), and a dominant negative of the
disease. Both types of interactions (soluble and direct)
Stat3 gene (Stat3b), these investigators were able to de-
may be potential targets for novel therapeutic approaches
lineate an IL-6-signaling pathway from Jak2 to Stat3 to
for myeloma.15
the bcl-xl gene promoter in myeloma cells (Figure 2).
Utilizing these inhibitors to block activation of Stat3 in
Inhibiting IL-6 Signaling
U266 cells inhibited Bcl-xL expression, induced apopto-
IL-6 is a critical growth factor for B-cell growth and de-
sis, and overcame resistance to Fas-mediated apoptosis.
velopment.16 Although IL-6 is involved in normal B-cell
These findings provide evidence that Stat3 activation
development, overproduction of this cytokine is consid-
contributes to the pathogenesis of myeloma by prevent-
ered to be an important component of the pathogenesis
ing apoptosis and conferring a survival advantage. Inter-
and progression of myeloma.17 The most common source
rupting Stat3 signaling may be a potential target for thera-
of IL-6 in myeloma is believed to be the bone marrow
peutic intervention in myeloma.
stromal cells, suggesting that IL-6 is a paracrine rather
than an autocrine growth factor in myeloma.17 In light of
Myeloma and Cell Adhesion-Mediated
these findings, it is generally believed that inhibiting IL-
Drug Resistance
6 signaling may be a novel approach to the treatment of
Intercellular interactions have long been known to con-
myeloma. Theoretically, this could be accomplished by
tribute to tumor cell survival and progression.22 Recently,
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Figure 1. Two different forms of tumor-microenvironment interactions influence drug response in cancer.
A. Soluble form of tumor-microvenvironment interaction (IL-6).
B. Direct contact form of tumor-microvenvironment interaction (ECM).
Figure 2. Constitutive activation of STAT3 prevents apoptosis in human myeloma cells. From Dalton and Jove. Immunity, 1999.21
it has been recognized that cell-cell and/or cell-ECM (ex-
tive myeloma cell line RPMI 8226 known to express both
tracellular matrix) adhesion may regulate apoptosis and
VLA-4 (
) and VLA-5 (
) integrin FN receptors,
4
1
5
1
cell survival in a wide variety of tumor types.23 Damiano
was relatively resistant to the apoptotic effects of doxo-
and colleagues recently presented evidence that the
rubicin and melphalan when cells were pre-adhered to
integrin receptors on myeloma cells may be responsible
FN compared to cells exposed to drug while in suspen-
for the cell adhesion-mediated drug resistance (CAM-
sion media (Figure 3). Recent data demonstrates that
DR) observed when cells are adhered to fibronectin
adhesion to FN protects cells from DNA damage induced
(FN).24 These investigators showed that the drug-sensi-
by DNA intercalating agents (e.g. doxorubicin), alkylat-
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ing agents (e.g. melphalan), and radiation treatments.
III. ADVANCES IN THE BIOLOGY AND TREATMENT OF
More recently, Hazlehurst and colleagues demonstrated
MYELOMA BONE DISEASE
that the cyclin dependent kinase (cdk) inhibitor, p27kip1
James R. Berenson, M.D.*
is overexpressed when cells are adherent to FN and this
cdk inhibitor may play an important role in effecting the
Biology
CAM-DR phenotype.25 These findings provide evidence
The major clinical manifestations of multiple myeloma
that antagonists of cellular adhesion, or signaling events
are related to loss of bone. This bone loss often leads to
related to adhesion, may serve as a means of inducing
pathologic fractures, spinal cord compression, hypercal-
myeloma cell apoptosis or improving the efficacy of anti-
cemia and bone pain. As a result, these patients often
cancer therapy for myeloma.
require analgesics, radiation therapy and surgery to bone.
This enhanced bone loss occurs because of the stimula-
Conclusions
tion of the cells responsible for bone resorption, the os-
There are at least three different approaches that may be
teoclasts, and results from the interplay between the tu-
used to improve therapeutic outcome for patients with
mor cells, other nonmalignant cells in the bone marrow
myeloma: (1) enhancing the efficacy of currently avail-
microenvironment and the bone-resorbing osteoclasts.
able chemotherapeutic drugs by identifying and overcom-
Specific soluble factors have been identified in the bone
ing drug resistance mechanisms, (2) identifying new tar-
marrow from myeloma patients that stimulate osteoclasts.
gets that regulate cell survival and growth of myeloma
Recent studies have elucidated the specific factors in-
cells, and (3) developing means to enhance host immune
volved in osteoclast stimulation and the ways that the
response against myeloma cells. However, no matter what
different cell populations interact in the bone marrow to
new approach clinicians may choose to investigate, a thor-
produce enhanced bone resorption in these patients.
ough understanding of the biology of myeloma will be
necessary for developing effective therapeutic approaches.
Bone Resorbing Factors
Understanding the biological principles that regulate
Initially, Mundy and colleagues identified a number of
myeloma cell survival and growth will likely provide new
proteins known as OAFs, osteoclastic activating factors,
targets for therapy and perhaps improve the efficacy of
which were thought to be the proteins responsible for
already available myeloma treatments.
enhanced bone loss in myeloma patients. These factors
including lymphotoxin (tumor necrosis factor) (TNF) )
* Department of Medicine, Cedars-Sinai Medical Center, 8700
Beverly Blvd., Bev. Mod.-1, Room 120, Los Angeles CA 90048
Figure 3. Fibronectin-induced drug resistance.
* p < 0.05 by Student's T-test; effect reversed by addition of 4 and 15-blocking Ab. Damiano et al.24
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and interleukin 1
(IL-1)
were identified in the super-
show increased levels of this cytokine.3 The role of IL-
natants from cultures from myeloma cell lines and fresh
1 in resorbing bone has been shown in bone organ cul-
myeloma bone marrow in these early studies. However,
tures. Although inhibitors of this protein and its recep-
more recent studies have suggested that other factors are
tors are capable of inhibiting bone resorption generated
important in leading to the loss of bone among these pa-
by supernatants derived from myeloma bone marrow,
tients.
other groups have been unable to show that this cytokine
The role of lymphotoxin in myeloma bone disease
plays a role in myeloma bone disease.2 Fresh bone mar-
has been downplayed by more recent studies failing to
row plasma from myeloma patients does not show higher
find significant differences in the amount of this cyto-
levels of IL-1 , and antibodies to this protein do not
kine in supernatants derived from bone marrow cultures
change the ability of myeloma bone marrow plasma to
or fresh bone marrow plasma derived from myeloma pa-
induce osteoclast formation.
tients compared to controls. In addition, antibodies to
IL-6, which is mainly produced by nonmalignant
lymphotoxin do not reduce the bone resorbing activity
cells in the bone marrow of myeloma patients, is a cyto-
of fresh bone marrow plasma from myeloma patients.
kine capable of stimulating growth and preventing apopto-
Another tumor necrosis factor, TNF , is found at higher
sis of the malignant cells in myeloma patients. However,
levels in supernatants from these patients' bone marrow
it also has been shown to stimulate bone loss by inhibit-
cultures, and is capable of stimulating osteoclast forma-
ing bone formation and stimulating osteoclast formation
tion. Its effects are mediated by stimulation of the pro-
especially in the presence of IL-1 or the soluble IL-6 re-
teolytic breakdown of I B that leads to the release of
ceptor (sIL-6R). Stromal cells, osteoclasts and osteoblasts
NF B. This enhancer translocates into the nucleus where
are all major sources of IL-6 in the bone marrow mi-
it induces transcription of specific genes, some of which
croenvironment. Recent studies show that malignant cells
are involved in enhancing bone resorption. The impor-
from myeloma patients increase IL-6 production by os-
tance of NF B in bone resorption is supported by recent
teoblasts both by direct cell-to-cell contact and release
studies showing that NF B knockout mice show osteo-
of soluble factors. Since some studies report that malig-
petrotic bones.
nant cells from myeloma patients produce IL-1 and TNF
A recently identified receptor for activation of NF B,
and both of these cytokines stimulate IL-6 production by
a member of the TNF receptor family, and its ligand,
osteoblasts,9,10 either or both may be the soluble factor(s)
RANKL, has been shown to be key players in the devel-
involved in myeloma cell-induced release of IL-6 by os-
opment of osteoclasts. Unlike other soluble bone resorb-
teoblasts. Thus, these studies suggest the role of bone
ing factors, the activity of these molecules requires di-
cells not only in bone-related changes in these patients
rect cell-to-cell contact. It has been known for some time
but also in the promotion of growth and prevention of
that osteoclastogenesis requires the direct interaction of
apoptosis of the tumor cells themselves as mediated by
osteoblasts or stromal cells with osteoclasts. The identi-
IL-6.
fication of RANK expressed on the surface of osteoclasts
IL-11 stimulates osteoclastogenesis and inhibits bone
and RANKL on osteoblasts and stromal cells explains
formation.11 It has been shown to be produced by osteo-
how this interaction leads to osteoclast development. TNF
blasts, and present in culture supernatants of bone mar-
itself is capable of stimulating osteoblasts to increase
row cells from myeloma patients. This cytokine stimu-
expression of RANKL. Malignant plasma cells from
lates RANKL expression by osteoblasts. In addition, re-
myeloma patients have been recently shown to express
cent studies have shown that hepatocyte growth factor
RANKL so that it is possible that the tumor cells them-
(HGF) which is known to be produced by malignant
selves may directly stimulate osteoclast development in
plasma cells,12 may also induce IL-11 secretion by os-
the myeloma bone marrow environment. Importantly, a
teoblasts.13 Other cytokines such as IL-1 are capable of
soluble decoy receptor called osteoprotegerin (OPG) ex-
potentiating the effect of HGF on IL-11 secretion. High
ists that binds RANKL and prevents the binding of the
serum levels of HGF are associated with a poor progno-
ligand to RANK. In fact, animals lacking OPG show pro-
sis in myeloma patients.
found osteoporosis. It is the delicate balance between
It is clear that macrophage colony-stimulating fac-
soluble OPG and RANKL that determines the amount of
tor (M-CSF) is involved in early events in the develop-
bone loss. Because of its profound inhibitory effect on
ment of osteoclasts. Although this factor along with
bone loss, OPG is now being evaluated in early clinical
RANKL are all that is required for osteoclastogenesis to
trials.
occur, its role in myeloma bone disease remains unclear.
Despite the early findings of Mundy identifying IL-
Some studies show higher amounts of this factor in the
1 as one of the OAFs in multiple myeloma patients, its
serum of myeloma patients and its correlation with tu-
role in myeloma bone disease has become less clear. Su-
mor load.
pernatants from myeloma patients' bone marrow cultures
Recently, macrophage inhibitory protein-1
(MIP-
Hematology 2000
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1 ), has been identified as an important factor involved
osteocalcin level or higher ICTP concentrations predicts
in myeloma bone disease.2 Levels of this cytokine are
a shortened survival in myeloma. In a recent placebo-
elevated in the bone marrow of these patients. This chemo-
controlled randomized Finnish clinical trial involving oral
kine is capable of inducing osteoclast formation in vitro,
clodronate, higher baseline levels of the amino-terminal
and antibodies to this protein block the induction of os-
propeptide of type I procollagen (PINP), a product of
teoclast formation by fresh bone marrow plasma from
growing osteoblasts, ICTP and alkaline phosphatase (AP)
myeloma patients. In addition, this chemokine attracts
were associated with a worse survival. PINP and ICTP
and activates monocytes, and is a potent inhibitor of early
levels decreased dramatically during clodronate treatment.
hematopoiesis.
Similarly, treatment with oral risedronate reduced uri-
There is evidence for an increasing role of angio-
nary pyridinoline/creatinine and deoxypyridinoline/crea-
genesis in the pathogenesis of multiple myeloma. It is
tinine ratios as well as the bone formation markers AP
clear that vascular endothelial growth factor (VEGF) is
and osteocalcin plasma levels. Monthly administration
produced by malignant plasma cells, and the receptors
of intravenous pamidronate is also associated with a de-
that bind this factor are expressed on bone marrow stro-
crease in both bone resorption and bone formation mark-
mal cells.14 In fact, recent results show that VEGF in-
ers. In the Finnish clodronate trial, a decrease in these
creases IL-6 production by bone marrow stromal cells
markers during clodronate therapy was associated with a
from myeloma patients. This may indirectly lead to en-
better survival. In current clinical trials evaluating newer
hanced bone loss in these patients. Until recently, it was
bisphosphonates, it is being determined whether baseline
not clear that VEGF had any direct role in bone resorp-
values or changes in these markers predict for either the
tion. However, it has now been shown that VEGF can
development of new skeletal complications or whether
replace M-CSF in leading to early osteoclast develop-
these agents will be clinically effective in individual cases.
ment.15
There is some evidence to support this from two recent
studies. In breast cancer patients with lytic bone me-
Adhesion Molecules
tastases receiving the aminobisphosphonate pamidronate
The critical role of the integrin
in bone resorption
who normalized urinary N-telopeptide, a newer bone re-
v
3
has been demonstrated in several recent studies.16,17 Mice
sorption marker, levels, there was a reduction in the de-
lacking the 3 molecule show reduced bone resorption,
velopment of new fractures as well as progression of bone
and antibodies and blocking ligands to
reduce os-
metastases.25 In a recent study evaluating another nitro-
v
3
teoclastic activity.16,17 The
molecule binds to a trip-
gen-containing bisphosphonate, ibandronate, in myeloma
v
3
eptide, RGD, within the extracellular matrix. When this
patients, increased suppression of urinary bone resorp-
integrin is bound to specific extracellular matrix proteins
tion markers was associated with reduction in the devel-
that contain this peptide, a chain of events occurs within
opment of bony complications.26
the osteoclast that results in a cell that is actively resorb-
ing bone. Both the protein tyrosine kinase Src and an-
Treatment of Myeloma Bone Disease
other specific kinase, Pyk-2, are activated upon
bind-
Although analgesics, surgery and radiotherapy may ef-
v
3
ing to the matrix proteins. The pivotal role of Src in bone
fectively palliate patients with complications from my-
resorption is supported by the development of osteopetro-
eloma bone disease, these treatments do little to slow the
sis in mice without Src.18 These animals contain abnor-
progressive bone loss that occurs in these patients. Che-
mal osteoclasts and lack bone-resorbing activity. In ad-
motherapy may reduce tumor burden but has little im-
dition to osteoclasts,
has also been shown to be
pact on the underlying bone disease. The demonstration
v
3
present on tumor cells and endothelial cells.19,20 This
that bisphosphonates could reduce the skeletal compli-
molecule is expressed by neovascularized blood vessels
cations and effectively palliate the symptoms related to
and its expression by tumor cells correlates with inva-
bone disease in myeloma patients has resulted in a dra-
siveness. Thus, this integrin is important in bone resorp-
matic change for the better in the lives of these patients.
tion, angiogenesis, and tumor invasion.
Importantly, because these agents lack significant bone
marrow suppressive effects, they can be administered as
Bone Resorption Markers
an adjunct to other cytotoxic therapy. Recent laboratory
A variety of markers of bone resorption and formation
studies suggest that these drugs have potential anti-my-
have been used to assess bone disease in myeloma pa-
eloma effects, and this is supported by clinical studies
tients.21-24 Patients with multiple myeloma show the ex-
showing an improvement in the survival of some patients
pected increases in bone resorption markers such as C-
receiving these drugs. Newer more potent aminobis-
terminal telopeptide of type I collagen, pyridinoline and
phosphonates are in active clinical trials and offer the
deoxypyridinoline and decreases in bone formation mark-
potential to further reduce bone-related problems while
ers such as osteocalcin. In addition, a decrease in
improving the overall outcome of myeloma patients. In
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addition to the bisphosphonates, a number of other types of
ber of proteins including guanidine triphosphatases such
anti-bone resorptive agents are entering clinical trials.
as ras and rho. Specifically, the addition of geranyl-
geranylated derivatives rather than farnesylated com-
Bisphosphonates
pounds is able to overcome the apoptosis-inducing ef-
fects of aminobisphosphonates and statin derivatives.29
Pharmacology
In addition, a reduction in the production of the cytokine
Bisphosphonates are analogues of endogenous pyrophos-
IL-6 from myeloma bone marrow stromal cells exposed
phate in which a carbon atom replaces the central atom
to bisphosphonates has also been found.30,31 This cyto-
of oxygen.27 This carbon substitution makes these com-
kine is not only capable of stimulating bone resorption
pounds resistant to hydrolysis, and allows two additional
but also is an important growth factor and anti-apoptotic
chains of variable structure. One of these side chains usu-
factor in myeloma.7 Thus, reducing the availability of this
ally contains a hydroxyl moiety that allows high affinity
cytokine in the bone micro-environment by exposure to
for calcium crystals and bone mineral. The differences at
bisphosphonates may not only reduce bone loss but may
the other side chain produce marked differences in the
also have an antimyeloma effect. Recent animal studies
anti-resorptive potency of different bisphosphonates. In
have shown that the aminobisphosphonates also have
fact, the newer bisphosphonates, such as ibandronate and
potent anti-angiogenic activity. Anti-angigoenesis agents
zoledronic acid, show 10,000­100,000-fold more potency
such as thalidomide have recently been shown to be ef-
than do the older agents such as etidronate. These drugs
fective antitumor agents in myeloma.32 Thus, the anti-
have limited bioavailability (usually < 1%) and are also
angiogenic effect of the aminobisphosphonates may con-
poorly tolerated orally, with significant gastrointestinal
tribute to these drugs' antibone resorptive effect (see
toxicity, particularly esophagitis and esophageal ulcers.
above) as well as provide additional mechanisms by
The bisphosphonates are almost exclusively eliminated
which these drugs may have antimyeloma effects as well.
through renal excretion. Although significant nephrotox-
A new potential anti-tumor mechanism for these com-
icity can occur, this is clearly related to the drug dose
pounds was recently reported for aminobis-
and rate of intravenous infusion. Importantly, this renal
phosphonates.33 These drugs were shown to induce ex-
dysfunction results from the bisphosphonate backbone
pansion of
T cells in peripheral blood mononuclear
so that the newer, more potent bisphosphonates can be
cell cultures, and enhance cytotoxicity of malignant
administered at therapeutic doses more rapidly without
plasma cells in bone marrow cultures by these T lym-
significant risk of nephrotoxicity.
phocytes. Thus, there is increasing evidence that
Bisphosphonates preferentially bind to bones that
bisphosphonates, especially the nitrogen-containing com-
have high rates of bone turnover (i.e. undergoing increased
pounds, can lead to direct and indirect effects that result
bone resorption or formation). Thus, these agents are
not only in less bone loss but less tumor burden as well.
concentrated at the exposed bone surface that undergoes
In support of this, Epstein and colleagues34 have shown a
active remodeling. Once these drugs are integrated into a
reduction in both lytic bone metastases and improvement
region of bone that is not undergoing remodeling, the
in survival in severe combined immunodeficiency mice
bisphosphonates lose their ability to inhibit bone resorp-
implanted with fresh human myeloma bone marrow and
tion. As a result, continued administration of these agents
fetal bone after treatment with pamidronate. However,
is required to achieve an enduring reduction in bone re-
treatment with ibandronate in a murine model of my-
sorption in a patient with lytic bone disease.
eloma35 showed only a reduction in lytic bone disease,
without an impact on tumor burden.
Mechanisms of action
The inhibition of bone resorption occurs as a result of
Clinical studies in myeloma patients
the effect of these drugs on osteoclasts both directly and
Small, open-label trials involving bisphosphonates sug-
indirectly. Bisphosphonates were first shown to reduce
gested their potential role in these patients. The first two
osteoclast development from their precursors as well as
randomized double blind placebo-controlled trials in-
inhibit movement of osteoclasts to the bone surface where
volved use of daily oral etidronate (5 mg/kg/d)36 or
they would normally resorb bone. These drugs are also
clodronate (2,400 mg/d)37 in newly diagnosed patients
capable of inducing apoptosis of osteoclasts as well as
who also received oral melphalan and prednisone. Nei-
tumor cells from myeloma patients.28,29 Interestingly, this
ther of these trials showed any significant clinical ben-
has been shown to occur as a result of inhibition of the
efit with these less potent oral agents, although there were
mevalonic acid pathway particularly in nitrogen-contain-
fewer patients developing new lytic lesions in the
ing bisphosphonates.29 Interestingly, the statin drugs that
clodronate trial who received the bisphosphonate. Oral
lower cholesterol also block enzymes in this same path-
clodronate has also been the subject of another random-
way. Both types of drugs prevent prenylation of a num-
ized double blind trial from the Medical Research Coun-
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cil.38 In addition to their chemotherapy, 536 newly diag-
entered clinical trials. Very small doses of these agents
nosed multiple myeloma patients received either 1,600
effectively restore normocalcemia in patients with tumor-
mg of clodronate or placebo daily. Although fewer pa-
induced hypercalcemia.42,43 Recent results show the su-
tients treated with clodronate experienced severe hyper-
periority of zoledronic acid (4 or 8 mg) compared to
calcemia and vertebral as well as nonvertebral fractures,
pamidronate (90 mg) in reversing hypercalcemia of ma-
there were no differences in the time to first skeletal event
lignancy.44 Clinical evaluation of zoledronic acid and
or requirement for radiotherapy. The drug had no signifi-
ibandronate in bone metastases is in progress. Zoledronic
cant effect on pain except back pain at a single time point
acid can be given safely over several minutes and pro-
(24 months), and performance status was also unaffected
duces similar anti-resorptive effects, as assessed by bone
except at this same time point. Patients receiving
resorption marker, as 90 mg of pamidronate.45 Prelimi-
clodronate showed no difference in survival compared to
nary reports from a randomized phase II study compar-
the placebo group. Oral pamidronate (300 mg/d) was
ing monthly infusions of zoledronic acid (0.4, 2 or 4 mg
compared to placebo in 300 newly diagnosed myeloma
as a 5-minute infusion) compared to pamidronate (90 mg
patients also receiving intermittent oral melphalan and
as a 2-hour infusion) in 280 patients with lytic bone me-
prednisone, and had no effect on skeletal-related mor-
tastases (109 myeloma) have been reported.46 The pro-
bidity or survival.39 Several small open-label studies sug-
portion of patients with any SRE was lower (30-35%) in
gested the possible benefit of infusional pamidronate in
the 2-mg and 4-mg zoledronic acid and the pamidronate
myeloma patients. As a result, 392 myeloma patients with
groups compared to the 0.4 mg zoledronic acid group
Durie-Salmon Stage III multiple myeloma and at least
(46%). This phase II trial was not "powered" to show
one lytic lesion were randomized to receive monthly 4-
superiority of zoledronic acid compared to pamidronate.
hour infusions of either placebo or pamidronate (90 mg)
An ongoing, larger phase III randomized trial compares
for 21 cycles.40,41
higher doses of zoledronic acid to 90-mg pamidronate in
Patients were stratified prior to randomization ac-
multiple myeloma or breast cancer with lytic disease.
cording to the amount of prior antimyeloma therapy at
Ibandronate is another newer potent bisphosphonate. A
study entry: stratum 1, first-line chemotherapy; stratum
phase III placebo-controlled trial of 214 myeloma pa-
2, second-line or greater chemotherapy. Initial results after
tients with Durie Salmon Stage II or III was recently com-
nine cycles showed a marked reduction in the proportion
pleted.26 Patients either received monthly bolus injections
of patients having any skeletal event with pamidronate
of 2 mg of ibandronate or placebo injections in addition
treatment,40 and this difference was also observed for
to their antineoplastic therapy. Ninety-nine patients in
patients in both stratum 1 and stratum 2. Patients receiv-
each group were evaluable for efficacy. The mean num-
ing pamidronate also showed decreases in bone pain and
ber of events per patient year on treatment was similar in
no increase in analgesic usage or deterioration in perfor-
both groups (ibandronate 2.13 vs placebo 2.05). How-
mance status or quality of life, in contrast to placebo pa-
ever, in the subgroup of ibandronate-treated patients
tients. The results after an additional 12 cycles of ran-
showing a sustained reduction in bone resorption mark-
domized treatment continued to show that the proportion
ers, less SREs/year occurred. There was no difference in
of patients developing any skeletal event remains smaller
overall survival. Thus, this dose of ibandronate is inad-
in the pamidronate group.41 Among all 392 patients, there
equate to show significant effects on preventing skeletal
was no difference in overall survival between the
complications in myeloma.
pamidronate and placebo groups. However, patients re-
ceiving pamidronate in stratum 2 showed a median sur-
New Agents
vival of 21 months compared with 14 months for pla-
A number of new agents are entering clinical trials for
cebo patients. Thus, it is clear from this large random-
the treatment of metastatic bone disease. An oral
v
3
ized trial that intravenous pamidronate (90 mg), when
antagonist, the RGD peptidomimetic SD-7784, is enter-
administered monthly as an adjunct to chemotherapy,
ing a phase I trial in myeloma patients.
antagonists
v
3
results in a significant reduction in the skeletal compli-
have previously been shown to have potent anti-tumor
cations as well as palliation of symptoms among my-
and anti-osteoclastic effects in animal models.16,17,20 Thus,
eloma patients with lytic bone disease. It is unclear
these drugs have the potential to both reduce bone loss
whether this drug or other bisphosphonates will be simi-
as well as reduce tumor burden in myeloma patients. An
larly effective for patients without lytic bone disease or
OPG analog, an inhibitor of RANK-RANKL interaction,
prevent patients with solitary plasmacytoma from devel-
is now being evaluated in a clinical trial in patients with
oping multiple myeloma. Third-generation bisphos-
bone metastases. In addition, inhibitors of src activity
phonates (e.g., zoledronic acid and ibandronate), that
show marked anti-resorptive capability in animal mod-
appear to be more than 100 times more potent than sec-
els47 and may enter clinical trials soon. The cholesterol-
ond-generation aminobisphosphonates, have recently
lowering statin drugs have recently shown to increase
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bone formation in vitro and in vivo in rodent models,48
to extracellular matrix (ECM) proteins and bone marrow
possibly by their induction of bone morphogenetic pro-
stromal cells (BMSCs), as well as changes in cell adhe-
tein (BMP)-2 as well as their ability to induce apoptosis
sion molecule profile correlating with egress of tumor
of osteoclasts. Some studies have suggested that patients
cells into the peripheral blood (PB) in the context of pro-
receiving statins to lower serum cholesterol also show a
gressive disease and plasma cell leukemia (PCL).2 Ad-
reduced risk of developing fractures. The apoptotic-in-
hesion molecules not only localize tumor cells within the
ducing effect of statins has also been shown to occur in
BM microenvironment but also have multiple functional
myeloma cells in vitro through their blockage of the
sequelae. Adherence to BMSCs confers resistance to
mevalonic acid pathway.29 Whether either the bone-en-
apoptosis,3 and agents that block adhesion, i.e.
hancing or anti-tumor effects of statins is clinically ob-
bisphosphonates, can confer sensitivity to treatment.
served in myeloma patients is being assessed in clinical
Adherence of tumor cells to BMSCs upregulates IL-6
trials. New attempts are underway to encourage new bone
transcription dependent in part on the transcription fac-
formation using BMPs, gene therapy and mesenchymal
tor NFkB, as well as IL-6 secretion within BMSCs,4 and
stem cells.49 Thus, the potential exists to not only halt the
also allows for tumor cell secretion of cytokines, i.e.,
relentless bone loss in myeloma patients but to repair their
transforming growth factor , which further enhances IL-
already damaged bones.
6 transcription and secretion in BMSCs.5 This is of cen-
tral importance since our studies have shown that IL-6 is
IV. NOVEL BIOLOGICALLY BASED THERAPIES
both a growth and survival factor for myeloma cells.6
FOR MYELOMA
Proteasome inhibitors are novel drugs that inhibit activa-
Kenneth C. Anderson, M.D.*
tion of NF B7; they induce apoptosis of myeloma cells
which are resistant to conventional therapy and impor-
Novel Therapeutic Targets
tantly, inhibit the NF B-dependent upregulation of IL-6
Multiple myeloma is incurable with current therapies, but
in BMSCs and related paracrine growth of adherent tu-
several recent biological advances have provided the
mor cells.8
framework for novel therapeutic strategies. First, mul-
We have shown that proliferation of myeloma cells
tiple lines of evidence suggest that the precursor cell in
triggered by IL-6 is mediated via the mitogen-activated
multiple myeloma is a cytoplasmic µ-positive B cell that
protein kinase (MAPK) cascade,9 suggesting therapeutic
has undergone antigen selection and somatic hyper-
strategies based upon blocking this pathway in tumor
mutation in the lymph node, but which has not yet un-
cells. Apoptosis triggered by gamma irradiation (IR), Fas,
dergone isotype class switching. Chromosomal translo-
and Dexamethasone (Dex) is mediated via distinct sig-
cations involving the immunoglobulin (Ig) switch region
naling cascades. For example, Dex (but not IR or Fas)-
are common, and multiple partner chromosomes have
induced apoptosis is mediated via activation of related
been described. Given that abnormalities in Ig gene rear-
adhesion focal tyrosine kinase (RAFTK).10 IL-6 is also a
rangement, IgH class switching, and DNA damage re-
survival factor for human myeloma cells, specifically
pair are hallmarks of myeloma, we have undertaken stud-
activating protein tyrosine phosphatase (SHP2) and
ies of Ku expression and function in human myeloma
thereby blocking the activation of RAFTK and related
cells.1 Ku is a heterodimer composed of Ku70 and Ku86
apoptosis in response to Dex.11 Blocking SHP2 activa-
subunits that binds with high affinity to altered DNA and
tion with small molecule SHP2 inhibitors may therefore
is essential for double stranded DNA break (DSB) repair
relieve this protective effect. Further delineation of these
and normal Ig V(D)J recombination. Our studies to date
pathways will derive strategies for triggering apoptosis,
have identified a 69 kD variant of Ku86 (Ku86v) in some
overcoming Dex resistance, and inhibiting survival sig-
myeloma cells, which neither binds DNA-dependent pro-
nals, which will provide the framework for related novel
tein kinase (DNA-PKcs) nor activates kinase activity and
treatment approaches.12
therefore may account for decreased DNA repair and in-
Our recent studies also suggest that adhesion of my-
creased sensitivity to radiation and chemotherapy; con-
eloma cells to BMSCs also upregulates vascular endot-
versely, Ku86 in myeloma cells confers resistance to
helial growth factor (VEGF) secretion by BMSCs and
therapy and may represent a therapeutic target.
myeloma cells. Therefore, in addition to examining the
Myeloma cells home to the bone marrow (BM) mi-
effect of VEGF on BM angiogenesis, we are evaluating
croenvironment, where excess plasma cells characteris-
whether VEGF is a growth and/or survival factor for
tic of this disease accumulate. We have demonstrated
myeloma cells. Preliminary studies suggest that VEGF
mechanisms whereby tumor cells specifically adhere both
induces MAPK activation and proliferation of some my-
eloma cells, and that VEGF receptor inhibitors block pro-
liferation of tumor cells and may therefore be useful clini-
* Dana-Farber Cancer Institute, 44 Binney Street, Boston MA
cally. This increase in VEGF may in part account for in-
02115
Hematology 2000
159

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creased angiogenesis in human myeloma BM. Based upon
testing of Thal and the IMiDs in a new treatment para-
its anti-angiogenic activity, thalidomide (Thal) was re-
digm to target both the tumor cell and the microenviron-
cently used very successfully to treat patients with my-
ment, overcome classical drug resistance, and achieve
eloma, even those refractory to conventional therapy.13
improved outcome in this presently incurable disease.
Although Thal may be acting in myeloma as an anti-an-
giogenic agent, there are multiple other potential mecha-
Immune-Based Strategies
nisms of action of Thal and/or its in vivo metabolites.14
Although high response rates can be achieved using high-
First, Thal may have a direct effect on the myeloma cell
dose therapy followed by stem cell grafting, the majority
and/or BMSC cell to inhibit growth and survival. For
of patients are destined to relapse and few, if any, are
example, free radical-mediated oxidative DNA damage
cured. Major obstacles to cure are the excessive toxicity
may play a role in the teratogenicity of Thal and may
noted after allografting in myeloma, contaminating tu-
also have anti-tumor effects. Second, adhesion of my-
mor cells in autografts, and most importantly, the persis-
eloma cells to BMSCs both triggers secretion of cyto-
tence of minimal residual disease (MRD) after high-dose
kines that augment myeloma cell growth and survival and
therapy followed by either allogeneic or autologous stem
confers drug resistance; thalidomide modulates adhesive
cell transplantation. In this context, we are developing
interactions and thereby may alter tumor cell growth,
improved strategies to treat MRD after high-dose therapy
survival, and drug resistance. Third, cytokines secreted
followed by allogeneic or autologous stem cell grafting.
into the BM microenvironment by myeloma and/or
Most importantly, we are developing multiple approaches
BMSCs, such as IL-6, IL-1 , IL-10 and TNF , may aug-
for the generation and enhancement of allogeneic and
ment myeloma cell growth and survival, and Thal may
autologous anti-myeloma immunity in vitro and in ani-
alter their secretion and bioactivity. Fourth, VEGF and
mal models. Based upon these studies, we are designing
basic fibroblast growth factor (bFGF)-2 are secreted by
clinical trials that couple our treatments to achieve MRD
myeloma and/or BMSCs and may play a role both in
with these novel immune-based therapies post transplant
tumor cell growth and survival, as well as BM angiogen-
in an attempt to achieve long-term disease-free survival
esis. Given its known anti-angiogenic activity, Thal may
and potential cure of multiple myeloma.
inhibit activity of VEGF, bFGF-2, and/or angiogenesis
in myeloma. Finally, Thal may be acting against myeloma
Allografting
via its immunomodulatory effects, such as induction of a
We have carried out high dose therapy followed by T
Th1 T cell response with secretion of interferon gamma
(CD6) cell-depleted allografting using histocompatible
(IFN- ) and IL-2. Understanding which of these mecha-
sibling donors in 61 patients with myeloma whose dis-
nisms mediate anti-myeloma activity will be critical both
ease remained sensitive to conventional chemotherapy.17
to optimally define its clinical utility and to derive ana-
This included 39 men and 22 women with median age of
logues with enhanced potency and fewer side effects.
44 (32-55) years. Most patients presented with advanced
Already two classes of Thal analogues have been reported,
stage myeloma. The majority of patients achieved either
including phosphodiesterase 4 inhibitors that inhibit
complete (28%) or partial (57%) response; importantly,
TNF but have little effect on T cell activation, and oth-
only 17% patients developed > grade 2 graft-versus-host
ers that are not phosphodiesterase inhibitors but do mark-
disease (GVHD), and the transplant-related mortality was
edly stimulate T cell proliferation as well as IFN- and
only 5%. Therefore we have shown that allografting can
IL-2 secretion.15 In recent studies, we have delineated
be done safely in myeloma. Indeed in our Center the over-
mechanisms of anti-tumor activity of Thal and its potent
all and progression-free survival of allograft and autograft
analogues (immunomodulatory drugs, IMiDs).16 Impor-
recipients is equivalent, with approximately 40% patients
tantly, these agents act directly, via inducing apoptosis
surviving at 3 years. However, only 20% patients are dis-
or G1 growth arrest, in myeloma cell lines and patient
ease free at > 4 years posttransplant. Excitingly, data from
myeloma cells that are resistant to melphalan, doxorubi-
our centers and others unequivocally demonstrate that
cin, and Dex. Moreover, Thal and the IMiDs enhance the
donor lymphocyte infusions (DLI) mediate a graft-ver-
anti-myeloma activity of Dex and, similar to Dex,
sus-myeloma effect (GVM) which can effectively treat
apoptotic signaling triggered by Thal and the IMiDs is
relapsed myeloma post allografting.18,19 Unfortunately
associated with activation of RAFTK. Most recent stud-
GVHD is a frequent cause of morbidity and mortality
ies suggest that treatment with these drugs augments their
after DLI. However, at our Myeloma Center five of seven
adherence to BMSCs and fibronectin, but abrogates the
patients who had relapsed post-CD6-depleted allograft-
upregulation of IL-6 and VEGF induced by tumor cell
ing responded (including three complete responses) to
binding. Finally, these drugs appear to upregulate natu-
CD4+ T cell enriched DLI, in some cases in the absence
ral killer cell-mediated killing of myeloma cells. These
of GVHD. This raised the possibility that distinct T cell
studies establish the framework for the development and
clones may be mediating GVM versus GVHD. Given the
160
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high response rates but inevitable relapses observed in
We are also attempting to generate and expand anti-my-
the setting of allografting for myeloma, we are now test-
eloma specific autologous T cells ex vivo for adoptive
ing in a clinical protocol whether CD4+ DLI at 6 months
immunotherapy of MRD in the patient post autotrans-
post-CD6-depleted allografting may mediate GVM,
plant. It is now possible to clone the gene for the patient's
which will effectively treat MRD and thereby improve
specific idiotypic protein, use computer programs to iden-
outcome. To date 21 patients have undergone CD6-de-
tify gene sequences encoding for peptides predicted to
pleted allografting, 18 of whom developed only grades
be presented within the groove of Class I HLA of a given
0­1 GVHD. Eleven of these 18 patients are > 6 months
patient's HLA type, and expand peptide specific T cells
posttransplant and have received CD4+ DLI. Eight of the
ex vivo.25 A similar strategy can be used to expand T
11 patients who received DLI demonstrated further re-
cells against peptides within shared antigens that are
sponse (including four complete responses), suggesting
overexpressed on myeloma cells, such as telomerase cata-
the potential of DLI to treat MRD. Therefore our studies
lytic subunit (hTERT),26 Muc-1,27 or CYP1B1.28 Strate-
already suggest that GVM can be adoptively transferred
gies are also being tested to enhance the immunogenic-
in this fashion. We are also examining T cell repertoire,
ity of the whole tumor cell. Our laboratory studies have
based upon V
T cell receptor gene rearrangement, to
shown that autologous T cells do not proliferate to the
identify those clonal T cells associated with GVM and
patients' own tumor cells as targets in an autologous
their target antigens on tumor cells.20,21 Already we have
MLR. However, CD40 activation of myeloma cells
shown that T cells mediating GVM can target idiotypic
upregulates Class I and II HLA, costimulatory, GRP94,
antigens and are presently identifying other target anti-
and other molecules; and CD40 activated myeloma cells
gens. The goal of these studies is to characterize, isolate,
trigger a brisk autologous T cell response.29 T cells can
and expand GVM T cell clones for antigen-specific adop-
therefore be harvested from myeloma patients before
tive immunotherapy.
autografting, expanded ex vivo using CD40-activated
autologous myeloma cells as stimuli, and given as adop-
Autografting
tive immunotherapy to treat MRD post transplant. Fi-
Although randomized studies convincingly demonstrate
nally, we are developing and examining the clinical util-
a survival advantage for myeloma patients treated with
ity of a variety of myeloma vaccines. First, based upon
high-dose therapy and autografting compared to those
our observation that CD40-activated myeloma cells trig-
receiving conventional chemotherapy, this treatment is
ger a brisk autologous T cell response, we will examine
not curative. Two sites of MRD contribute to the failure
the utility of vaccinations of patients with autologous
of autografting: MRD in the autograft and MRD in the
CD40 activated tumor cells. Second, based upon our dem-
patient post myeloablative therapy. At our Center we have
onstration of the expression of Muc-1 core protein on
to date carried out high dose therapy and stem cell
freshly isolated myeloma cells,27 we will construct and
autografting in 105 patients who presented with advanced
evaluate two vaccines: recombinant vaccinia virus con-
stage myeloma but whose disease remained sensitive to
taining the Muc-1 gene and autologous dendritic cells
chemotherapy. As in our allografting experience, the
(DCs) transduced using adenoviral vectors with Muc-1.
majority of patients responded, including 30% complete
Excitingly, we have recently shown that myeloma cells
and 62% partial responses. However, none of these pa-
can be fused to DCs and that the use of the myeloma
tients are cured. We have produced monoclonal antibod-
cell-DC fusion as an antigen presenting cell presents the
ies in the laboratory that have been used to deplete tumor
entire myeloma cell as foreign. In a syngeneic murine
cells from myeloma autografts.22 We have also evaluated
myeloma model, vaccinations with myeloma cell-DC
CD34 selection techniques to select normal hematopoi-
fusions, but not with either myeloma cells or DCs alone,
etic progenitor cells within autografts.23 However, these
demonstrated both protective and therapeutic efficacy.
methods deplete only 2­3 logs of tumor cells, and > 50%
Most importantly, we have shown that patient myeloma
autografts still contain MRD. Based upon our laboratory
cells can be fused to autologous DCs, which are readily
data that myeloma cells express Muc-1 and adenoviral
isolated from either patient bone marrow and peripheral
receptors, we have specifically transduced tumor cells
blood,30 and that autologous myeloma cell-DC fusions
within myeloma autografts with the thymidine kinase (tK)
can trigger specific cytolytic autologous T cell responses
gene using an adenoviral vector with a tumor selective
in vitro.31 We will therefore translate these findings to
(Muc-1) promotor, followed by purging tumor cells ex
the bedside in clinical trials of myeloma-DC fusion vac-
vivo by treatment with ganciclovir.24 Pilot studies sug-
cines to assess in vivo myeloma-specific T and B cell
gest that > 6­7 logs of tumor cells can be purged under
responses as well as clinical efficacy. Ultimately, vacci-
conditions that do not adversely affect normal hemato-
nations will be coupled with adoptive immunotherapy in
poietic progenitor cells, setting the stage for a clinical
an attempt to treat MRD post autografting and thereby
trial of adenoviral purging prior to autotransplantation.
improve outcome.
Hematology 2000
161

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