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Abstract
This review discusses the evolution of novel diagnostic and treatment strategies for multiple myeloma based upon increased understanding of basic disease pathogenesis. Although myeloma has remained an incurable illness to date, these new developments will derive treatments to improve outcome and achieve eventual cure.
In Section I, Dr. Kyle reviews the results of current therapy for multiple myeloma, including high dose therapy and stem cell transplantation which have proven to achieve improved response rates, event-free, and overall survival. Supportive therapy, such as erythropoietin to treat disease-related anemia, and methods of prophylaxis against infection, which both lessen toxicities of treatment and improve quality of life for patients, are also addressed.
In Section II, Dr. Dalton with Drs. Landowski, Shain, Jove and Hazlehurst discusses mechanisms of drug resistance in myeloma, with emphasis on novel treatment approaches to prevent development of drug resistance and to overcome drug resistance. Laboratory studies delineating mechanisms whereby myeloma cells resist drug-induced apoptosis provide the framework for related treatment protocols for patients with refractory disease.
In Section III, Dr. Berenson reviews the management of complications in bone, which occur in the majority of patients with myeloma and are the major cause of decreased quality of life. New insights into the mediators of bone resorption and new bone formation in the marrow milieu have already derived effective bisphosphonate therapy. These drugs not only reduce bone complications and related pain, thereby improving quality of life, but also may have intrinsic anti-tumor activity by virtue of inducing tumor cell adherence to marrow, reducing interleukin-6 secretion, inducing tumor cell apoptosis, or inhibiting angiogenesis.
In the last section, Dr. Anderson explores the potential for future
therapies which offer great promise to improve patient outcomes. First, drugs
which alter the marrow microenvironment include thalidomide and its derivative
immunomodulatory drugs, which act directly on tumor cells to induce apoptosis
or G1 growth arrest, alter tumor cell adhesion to marrow stroma, inhibit
angiogenesis, and trigger a cellular anti-tumor response. The proteasome
inhibitors both act directly on tumor cells and also inhibit the transcription
factor NF
B-dependent upregulation of IL-6 secretion triggered by tumor
cell adhesion. Second, delineation of both growth and apoptotic pathways has
derived novel treatment strategies. Third, the preclinical basis and early
clinical trial results using vaccination and adoptive immunotherapy to harness
autoimmune and alloimmune anti-myeloma responses are presented. This review
sets the stage for an evolving new biologically based treatment paradigm in
myeloma targeting both the tumor and its microenvironment to improve outcome
and achieve eventual cure.
I. Current Therapy of Myeloma
Robert A. Kyle, M.D.*
Although most patients with multiple myeloma (MM) have symptomatic disease at diagnosis and require therapy, some are asymptomatic and should not be treated. If there is doubt about beginning therapy, it is best to reevaluate the patient in two or three months and delay therapy until progressive disease is evident.
If the patient is younger than 70 years, the physician should discuss the possibility of an autologous peripheral blood stem cell transplant with the patient. Hematopoietic stem cells should be collected before the patient is exposed to alkylating agents. Chemotherapy is the preferred initial treatment for symptomatic MM in persons older than 70 years or in younger patients in whom transplantation is not feasible.
Peripheral blood stem cells are preferable to bone marrow transplantation because engraftment is more rapid and there is usually less contamination of the infused cells with tumor cells. The absolute number of CD34+ cells is the most reliable and practical method for determining the efficacy of the stem cell product. Autologous peripheral stem cell transplantation is applicable for more than half of patients with MM. The two major shortcomings are: 1) Eradication of myeloma from the patient rarely occurs even with large doses of chemotherapy and/or radiation and 2) autologous peripheral blood stem cells are contaminated by myeloma cells or their precursors. Fortunately, mortality from autologous transplantation is only 1-2% if patients are appropriately selected.
Most physicians initially treat the patient with vincristine and
doxorubicin (Adriamycin) given IV for 96 hours and dexamethasone orally (VAD)
for 3-4 months to reduce the number of tumor cells. Dexamethasone with or
without thalidomide is being evaluated for initial therapy. Peripheral blood
stem cells are collected following granulocyte colony-stimulating factor
(G-CSF) with or without high-dose cyclophosphamide. One can then proceed with
the transplant following high-dose chemotherapy and/or total body irradiation
(TBI) followed by infusion of the peripheral blood stem cells. The other
choice is to treat the patient with alkylating agents after stem cell
collection until a plateau is reached and then give the patient
alpha-2-interferon (
2IFN) or no therapy until early relapse.
At that time, the patient is given highdose melphalan with or without total
body radiation and the previously collected peripheral blood stem cells are
infused. Early or late transplantation are reasonable options. In one study,
185 patients were treated with three or four courses of VAD and then
randomized to highdose chemotherapy and autologous stem cell transplantation
or to conventional chemotherapy. There was no difference in median survival of
the two groups (65 vs. 64 months). The main advantage of early transplantation
was a shorter period of
chemotherapy.1
A randomized trial by the French Myeloma Group compared high-dose chemotherapy followed by autologous bone marrow transplantation with conventional chemotherapy in 200 previously untreated myeloma patients under the age of 65 years.2 Data was analyzed on an intention-to-treat basis. Twenty-five percent of the patients randomized to transplantation did not receive a transplant. Response rate (81 % vs 57%) and complete response rate (22% vs. 5%) were superior in the transplant group. The 5-year event-free survival (28% vs 10%) and overall survival (52% vs 12%) were superior in the transplant group. It must be kept in mind that patient selection plays an important role in response and survival. In one report, 77 patients with MM who fulfilled the criteria for transplant (age < 66 years, Stage II or III, good performance status and disease responsive to initial chemotherapy) but who were treated with conventional chemotherapy had a survival of 5 years, which is similar to that seen in autologous stem cell transplantation.3
In a series of 177 patients < 75 years of age with IgG myeloma, C-VAMP followed by high-dose chemotherapy with or without stem cell rescue and maintenance interferon were reported. The median survival was 4.9 years. Those with ß2-microglobulin <2.7 mg/L and those < 52 years of age had a more favorable response.4
The role of TBI in the preparative regimen is controversial. In a comparison of melphalan, 140 mg/m2 plus TBI vs melphalan 200 mg/m2, there was no difference in response rate, event-free survival and overall survival. However, the toxicity of melphalan 200 mg/m2 was significantly lower than melphalan plus TBI.5 Consequently, many have discontinued TBI and give only melphalan, 200 mg/m2 for the preparative regimen.
The role of double or tandem autologous stem cell transplants is controversial. In an uncontrolled series of 231 newly diagnosed MM patients who received a second transplant, 51 % achieved complete response and 95% had a complete or partial response. The authors felt that the double transplant extended both event-free and overall survival even in patients with unfavorable cytogenetics and ß2-microglobulin values.6 In a randomized trial of 400 patients from France, there was no difference in event-free or overall survival between single and double autologous stem cell transplants when evaluated at two years. The two groups were similar from the standpoint of age, gender, stage, Ig isotype, ß2-microglobulin value, C-reactive protein level and bone marrow plasmacytosis. The complete response rate was 32% with a single transplant and 33% with a double transplant. At 2 years, the event-free survival was 54% vs. 57% while the overall survival was 71% vs 67%.7 In a subsequent evaluation, patients with a low ß2-microglobulin value at diagnosis appeared to have better results with the double transplant.
Almost all patients will relapse following an autologous stem cell
transplant. A preliminary analysis of a randomized trial of 85 patients with
MM who were treated with high-dose melphalan and autologous bone marrow
transplantation followed by 2-IFN maintenance therapy
suggested both a relapse-free survival and overall survival benefit. However,
the final analysis of this trial demonstrated no significant difference in
relapse-free or overall survival among patients randomized to maintenance
therapy with
2-IFN.8
Idiotype-treated autologous dendritic cells are being used to prolong response
duration.9
In an effort to prolong survival, highly purified CD34+ cells did not influence the achievement of clinical or molecular complete remission or remission duration or overall survival.10 Thus, tumor cell purging does not appear to be beneficial.
The use of 2-IFN for more than six months following
autologous stem cell transplantation resulted in delay of platelet
recovery.11 Patients refractory to VAD had the same overall
survival as those who responded to
VAD.12 In a
retrospective study, high-dose therapy for newly diagnosed myeloma resulted in
prolongation of survival for patients < 60 years old when compared to
historic controls treated with chemotherapy (61 vs 46
months).13
Allogeneic Bone Marrow Transplantation
The major advantage of allogeneic transplantation is that the graft
contains no tumor cells that can lead to a relapse. Unfortunately, over 90% of
patients with multiple myeloma are ineligible because of their age, lack of an
HLA-matched sibling donor or inadequate renal, pulmonary or cardiac function.
Furthermore, there is a mortality of at least 25% at present.
In a report of 266 patients from the European Blood and Bone Marrow Transplantation registry, 51% obtained a complete response. The overall treatment mortality rate was approximately 40%. The actuarial survival was 30% at 4 years and 20% at 10 years.14
It is obvious that the mortality rate for allogeneic transplantation must be reduced before it can assume a major role in the treatment of multiple myeloma. The use of a "mini-allo" transplant15 or depletion of T cells in an effort to reduce transplant mortality are promising approaches. Graft-versus-myeloma effect has been noted after donor peripheral blood mononuclear cells were given for relapse following allogeneic transplantation. Eight of 13 patients with relapsed myeloma following an allogeneic bone marrow transplantation responded to donor lymphocyte infusions.16
Chemotherapy
Various combinations of therapeutic agents have been used because of the
shortcomings of melphalan and prednisone. Melphalan and prednisone produces an
objective response in 50-60% of patients. In an overview of individual data
from 4,930 persons from 20 randomized trials comparing melphalan and
prednisone with a variety of combinations of chemotherapeutic agents, the
response rates were higher with combination chemotherapy (60%) than melphalan
and prednisone (53%) (p < 0.00001). However, there was no significant
difference in overall survival and there was no evidence that any group of
patients benefited from receiving combination
chemotherapy.17
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
myelodysplastic syndrome or acute leukemia. The possible benefit of
maintenance therapy with 2-IFN following conventional
chemotherapy is controversial due to conflicting results and frequency of
undesirable side effects. In a large meta-analysis Wheatley reported a
survival benefit in both induction (p = 0.05) and maintenance (p = 0.03) with
an increase in median response duration of six months in both
settings.18
Patients should be monitored closely during the plateau phase and the same
chemotherapy regimen should be reinstituted if relapse occurs after six
months. The use of prednisone, 50 mg orally every 48 hours, appears to prolong
the plateau state as well as the overall
survival.19
Treatment of Refractory Multiple Myeloma
Patients who are initially refractory or who become refractory to
alkylating agent therapy have a modest response rate to subsequent
chemotherapy and a limited survival. The highest response rates in patients
with MM resistant to alkylating agents have been with vincristine, doxorubicin
and dexamethasone (VAD). Vincristine and doxorubicin are given by continuous
infusion for four days and dexamethasone (40 mg daily) is given on days 1-4,
9-12, and 17-20 each month. Dexamethasone is often given only on days 1-4 in
even-numbered cycles because of toxicity. Dexamethasone can be given as the
only therapeutic agent since it accounts for approximately 80% of the effect
of VAD. Vincristine (0.4 mg) and doxorubicin (9 mg/m2) as a rapid
intravenous infusion daily for 4 days plus dexamethasone (40 mg) produced an
objective response in 67% of 134 previously untreated myeloma patients. This
suggests that 4-day infusion of vincristine and doxorubicin is
unnecessary.20
Intravenous pulse methylprednisolone (2 g three times weekly) is helpful for
patients with pancytopenia and refractory disease. We find fewer side effects
from this approach than with
dexamethasone.21
Vincristine (2 mg), carmustine (BCNU; 30-40 mg) and doxorubicin (30-40 mg)
intravenously on day 1 and prednisone daily for 5 days every 3 to 4 weeks
produces benefit in about one-third of patients. Thalidomide produced
responses in 32% of 84 patients with previously treated, progressive MM. After
12 months of follow-up, 22% of patients remained event free and 58% were
alive.22
Thalidomide was given in an initial dosage of 200 mg daily and gradually
increased to 800 mg daily. Constipation, weakness or fatigue, sleepiness, skin
rash and peripheral neuropathy were undesirable side effects. In the majority
of patients, response occurred within six weeks and with only 400 mg of
thalidomide daily. The use of thalidomide in conjunction with dexamethasone is
being explored.
Supportive Care
Renal failure
Approximately 20% of patients with MM have a creatinine level 
2 mg/dL
at diagnosis. The two major causes of renal insufficiency are "myeloma
kidney" and hypercalcemia. Dehydration, infection, nonsteroidal
anti-inflammatory agents and roentgenographic contrast media may contribute to
acute renal failure. Amyloid deposition occurs in 10% of patients who have MM
and often causes nephrotic syndrome, renal insufficiency or congestive heart
failure.
Maintenance of a high urine output (3 L/day) is important for preventing renal failure. Prompt treatment of hypercalcemia as well as correction of dehydration and electrolyte imbalance is crucial. Alkalinization of the urine may be useful.
Acute renal failure should be treated with appropriate fluid and electrolyte replacement and VAD in an effort to reduce the tumor mass as quickly as possible. A trial of plasmapheresis in younger patients with acute renal failure is a reasonable approach.23 Hemodialysis or peritoneal dialysis is necessary in the event of symptomatic azotemia.
Anemia
Anemia occurs in almost all patients during the course of MM. Fifty to
sixty percent of patients respond to
erythropoietin.24,25
Improvement in the patient's quality of life and an improved sense of
well-being also
occurs.26
Infection
Patients should receive pneumococcal and influenza vaccinations despite
their suboptimal antibody response. Prompt and appropriate therapy of
bacterial infections is essential. Prophylactic daily oral penicillin often
benefits patients with recurrent pneumococcal infections. Since many
infections occur in the first two months after instituting chemotherapy, the
use of daily oral trimethoprim/sulfamethoxazole is
helpful.27
Intravenously administered gamma globulin may be beneficial for patients with
recurrent infections, but it is inconvenient and very expensive.
Spinal cord compression
This complication should be suspected in patients with severe back pain who
develop weakness or paresthesias of the lower extremities or bladder or bowel
dysfunction. Magnetic resonance imaging or computed tomography must be done
immediately. An MRI is particularly useful in demonstrating extramedullary
plasmacytoma. Radiation therapy and dexamethasone are usually effective, and
surgical decompression is rarely necessary.
Hyperviscosity
This is characterized by oral or nasal bleeding, blurred vision,
paresthesias, headache or congestive heart failure. It may occur from high
concentrations of IgA or, rarely, IgG. The serum viscosity levels do not
correlate well with the symptoms or clinical findings. Consequently, a
decision to perform plasmapheresis depends on the symptoms and changes in the
ocular fundus. Plasmapheresis promptly relieves the symptoms and should be
done regardless of the viscosity level if the patient has signs or symptoms of
hyperviscosity.28
Emotional support
All patients with MM need substantial and continuing emotional support. The
physician's approach must be positive in emphasizing the potential benefits of
therapy. It is reassuring for patients to know that some survive for 10 years
or more. It is vital that the physician caring for patients with MM has the
interest and capacity to deal with an incurable disease over the period of
years with assurance, sympathy and resourcefulness.
II. Determinants of Drug Response and Drug Resistance in Multiple Myeloma
William S. Dalton, M.D., Ph.D.,* Terry Landowski, Kenneth Shain, Richard Jove and Lori Hazlehurst
Effective therapy for multiple myeloma began in the early 1960s with the development of alkylating agents, in particular, melphalan and cyclophosphamide.1 Even today, the combination of oral melphalan and prednisone is considered to be the mainstay of treatment for myeloma producing responses in approximately 50-60% of patients. During the past four decades, various drugs have been found to be effective for the treatment of myeloma; most of these drugs belong to the following pharmacologic classifications: alkylating agents (melphalan, cyclophosphamide, BCNU), topoisomerase II inhibitors (doxorubicin and etoposide), glucocorticoids (prednisone and dexamethasone), and the anti-tubulin agent, vincristine. With the exception of the glucocorticoids, most of these agents are relatively ineffective as single agents and are best administered as a combination of agents. Since the introduction of melphalan and prednisone, several drug combinations have been investigated. A popular combination of drugs proven to be effective is the M2 protocol consisting of vincristine, carmustine, melphalan, cyclophosphamide and prednisone (VBMCP).2 While this combination produces higher response rates than melphalan and prednisone, there is only a marginal improvement in overall survival that appears to occur chiefly for patients with a poor prognosis.3 Similar observations have been made for other drug combinations including vincristine, melphalan, cyclophosphamide, prednisone (VMCP) alternating with vincristine, carmustine (BCNU), doxorubicin and prednisone.4 The combination of VAD (vincristine, doxorubicin, and dexamethasone) produces responses in 60-70% of patients who become resistant to alkylating agents, but ultimately patients will develop resistance to all known chemotherapy regimens.5 The problem of drug resistance is a major obstacle to curing myeloma and understanding factors that determine drug response and the development of drug resistance should prove useful in developing means of preventing or overcoming this problem.6
Substantial progress has been made in the last decade in the identification of cellular mechanisms that confer clinical drug resistance in myeloma. Research to date has chiefly focused on intrinsic cellular mechanisms of drug resistance; in other words, mechanisms that reside within the myeloma cell itself. Examples of intrinsic cellular drug resistance include the following: (a) reduction of intracellular drug concentration due to the overexpression of membrane pump proteins such as P-glycoprotein, (b) altered drug metabolism or enhanced drug detoxification, (c) alterations in the drug target that reduce drug efficacy, and (d) enhanced cellular repair of drug-induced damage.6 Any one of these mechanisms could occur within a myeloma cell, and in fact, it is likely that a combination of these mechanisms occurs simultaneously within the same myeloma cell. Studies both in the laboratory and the clinic have demonstrated that eliminating or preventing a single mechanism of resistance, such as reduced intracellular drug concentration due to P-glycoprotein, selects for alternative mechanisms of drug resistance. Based on this evidence, attempting to overcome a single mechanism of intrinsic drug resistance is not likely to produce long-standing clinical remissions. In order to make substantial progress in the treatment of myeloma, it is important to develop a better understanding of the biological principles that govern myeloma cell survival and growth. Understanding these principles will allow investigators to develop therapeutic approaches that capitalize on the uniqueness of myeloma cells, thereby targeting the malignant cell and sparing normal cells.
Targeting Intrinsic Molecules and Pathways in the Myeloma
Cell
One approach that may prove to be successful in improving myeloma therapy
is the development of small molecules that inhibit or interrupt cellular
targets or pathways that regulate myeloma cell growth and survival. These
pathways may be intrinsic to the myeloma cell itself, such as altered signal
transduction pathways due to Ras mutations. Mutations in the Ras family of
genes are relatively common in myeloma patients. Neri and colleagues first
reported in 1989 that 32% of patients with myeloma had Ras
mutations.7 The
most frequent mutation occurred in the N-Ras gene, particularly at codon 61.
The second most frequent mutation was found at codon 12 for K-Ras. The
frequency of Ras mutations increased from 27% at diagnosis to 46% with disease
progression. Interestingly, these investigators found that patients with
mutated Ras were less likely to respond to chemotherapy compared to patients
with wild-type Ras. In a more recent study of 346 newly diagnosed, untreated
patients, Liu et al reported a total incidence of 39% Ras
mutations.8
Patients with Ras mutations had a median survival of 2.1 years compared to 4.0
years for patients with wild-type Ras. These studies demonstrate that Ras
mutations adversely affect survival and may reduce response to chemotherapy;
therefore, targeting Ras signaling may be a novel therapeutic approach to the
treatment of myeloma.
One approach to the interruption of Ras signaling in the myeloma cell is to prevent Ras "processing," a requirement for the activation of Ras.9 The enzyme farnesyltransferase is responsible for transferring the farnesyl group from farnesyl diphosphate, a cellular chemical used in the synthesis of cholesterol, to certain proteins such as Ras. This process is important for Ras activation because it allows the protein to attach to the inner plasma cell membrane. Preventing Ras from going to the plasma membrane by blocking its farnesylation short-circuits oncogenic growth signals to the nucleus. Inhibition of the farnesylation process is a prime target for drugs aimed at preventing Ras activity.10 Recently, several drugs have been synthesized to inhibit protein farnesylation. These drugs are called farneslytransferase inhibitors (FTIs) and preclinical studies have shown them to be effective in tumors with Ras mutations. A phase II clinical trial using the Janssen FTI (R11 5777) has been approved by CTEP for the treatment of myeloma and should begin soon. A second approach to inhibiting Ras processing may be the use of amino-bisphosphonates. Shipman et al found that the bisphosphonate incadronate caused apoptosis in human myeloma cells by inhibiting the mevalonate pathway.11 This pathway is essential for the biosynthesis of sterols and long-chain isoprenoid lipids including farnesyl PPi and geranylgeranyl PPi. These latter two compounds serve as substrates for farnesyltransferase and geranylgeranyl transferase, respectively, and are transferred to small GTP-binding proteins such as Ras. Inhibiting isoprenylation of these critical proteins may induce apoptosis in myeloma cells.
Targeting Extrinsic Molecules or Pathways Involved in
Myeloma Cell Survival and Growth
In addition to the intrinsic molecules or pathways discussed
above, there are signal transduction pathways that rely on external
communication between the myeloma cell and its environment. Similar to the
intrinsic pathways, these extrinsic factors also regulate myeloma
cell survival and growth and are potential targets for novel therapy of
myeloma. For example, the primary source of interleukin 6 (IL-6) is from the
bone marrow stroma and not the myeloma cell
itself.12
Inhibiting IL-6 production, binding to its receptor, or downstream signaling
may block myeloma cell proliferation and result in apoptosis. Myeloma cells
express cell adhesion molecules that allow attachment and communication
between the myeloma cell and the bone marrow
microenvironment.13
This communication influences myeloma cell survival and growth; interrupting
cellular adhesion may induce programmed cell death and enhance the efficacy of
standard treatment of myeloma. In addition, it has recently been reported that
myeloma cell growth and dissemination may depend upon
angiogenesis.14
Interactions between the microenvironment and the myeloma cells may regulate
angiogenesis and stimulate myeloma growth and progression. Blocking
intercellular communications by blocking cell adhesion may represent a new
approach for the treatment of myeloma.
Figure 1 demonstrates two possible forms of interaction between myeloma cells and the bone marrow microenvironment. A soluble form of interaction is represented by the interaction between IL-6, a cytokine produced by the bone marrow stromal cells, and the myeloma cell. Interaction between the myeloma cell and the bone marrow stroma mediated by cell adhesion molecules represents a direct form of interaction. For example, the interaction between cell surface integrins and extracellular matrix components, such as fibronectin, may be involved in prolonging cell survival and dissemination of disease. Both types of interactions (soluble and direct) may be potential targets for novel therapeutic approaches for myeloma.15
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Inhibiting IL-6 Signaling
IL-6 is a critical growth factor for B-cell growth and
development.16
Although IL-6 is involved in normal B-cell development, overproduction of this
cytokine is considered to be an important component of the pathogenesis and
progression of
myeloma.17 The most
common source of IL-6 in myeloma is believed to be the bone marrow stromal
cells, suggesting that IL-6 is a paracrine rather than an autocrine growth
factor in
myeloma.17 In light
of these findings, it is generally believed that inhibiting IL-6 signaling may
be a novel approach to the treatment of myeloma. Theoretically, this could be
accomplished by inhibiting the production of IL-6, blocking the binding of
IL-6 to its cell surface receptor, or by blocking down-stream signaling events
within the myeloma cell
itself.12 One
approach to blocking IL-6 receptor activation is to generate IL-6 variants
that block the receptor in an inactive form. One IL-6 variant in particular,
called Sant7, is a super-antagonist found to be effective at blocking
IL-6.18 Preclinical
testing of this super-antagonist is ongoing in hopes that Sant7, or similar
agents, may soon be tested in the clinic (G. Ciliberto, personal
communication).
Another approach to inhibiting IL-6 is to interrupt the intracellular signaling associated with cytokine receptor activation. IL-6 induces intracellular signaling through multiple pathways, but one pathway utilized by IL-6 to influence cell survival and resistance to apoptosis is the JAK/STAT pathway. Engagement of cell surface cytokine receptors activates the Janus kinase (JAK) family of tyrosine kinases, which in turn phosphorylate and activate the cytoplasmic STAT (signal transducers and activators of transcription) proteins.19 Activated STATs dimerize upon activation by JAKs and translocate to the nucleus where they bind specific DNA response elements and regulate the expression of certain genes. Seven mammalian STAT family members have been cloned and share common structural characteristics. One particular STAT member, Stat3, has been associated with oncogenesis when it is constitutively activated.20 A recent report by Jove and colleagues demonstrated that Stat3 is constitutively activated in bone marrow mononuclear cells in patients with myeloma.21 In addition, Jove and co-workers demonstrated high levels of activated Stat3 in the myeloma cell line U266 known to produce and utilize IL-6 for survival. Moreover, this cell line is resistant to Fas-mediated apoptosis in spite of high levels of Fas receptor being expressed in U266 cells. Using specific inhibitors of the IL-6 receptor (Sant7), a Jak2 inhibitor (AG490), and a dominant negative of the Stat3 gene (Stat3b), these investigators were able to delineate an IL-6-signaling pathway from Jak2 to Stat3 to the bcl-xl gene promoter in myeloma cells (Figure 2). Utilizing these inhibitors to block activation of Stat3 in U266 cells inhibited Bcl-xL expression, induced apoptosis, and overcame resistance to Fas-mediated apoptosis. These findings provide evidence that Stat3 activation contributes to the pathogenesis of myeloma by preventing apoptosis and conferring a survival advantage. Interrupting Stat3 signaling may be a potential target for therapeutic intervention in myeloma.
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Myeloma and Cell Adhesion-Mediated Drug Resistance
Intercellular interactions have long been known to contribute to tumor cell
survival and
progression.22
Recently, it has been recognized that cell-cell and/or cell-ECM (extracellular
matrix) adhesion may regulate apoptosis and cell survival in a wide variety of
tumor types.23
Damiano and colleagues recently presented evidence that the integrin receptors
on myeloma cells may be responsible for the cell adhesion-mediated drug
resistance (CAM-DR) observed when cells are adhered to fibronectin
(FN).24 These
investigators showed that the drug-sensitive myeloma cell line RPMI 8226 known
to express both VLA-4 (4ß1) and VLA-5
(5ß1) integrin FN receptors, was relatively
resistant to the apoptotic effects of doxorubicin and melphalan when cells
were pre-adhered to FN compared to cells exposed to drug while in suspension
media (Figure 3). Recent
data demonstrates that adhesion to FN protects cells from DNA damage induced
by DNA intercalating agents (e.g. doxorubicin), alkylating agents (e.g.
melphalan), and radiation treatments. More recently, Hazlehurst and colleagues
demonstrated that the cyclin dependent kinase (cdk) inhibitor,
p27kip1 is overexpressed when cells are adherent to FN and this cdk
inhibitor may play an important role in effecting the CAM-DR
phenotype.25 These
findings provide evidence that antagonists of cellular adhesion, or signaling
events related to adhesion, may serve as a means of inducing myeloma cell
apoptosis or improving the efficacy of anti-cancer therapy for myeloma.
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Conclusions
There are at least three different approaches that may be used to improve
therapeutic outcome for patients with myeloma: (1) enhancing the efficacy of
currently available chemotherapeutic drugs by identifying and overcoming drug
resistance mechanisms, (2) identifying new targets that regulate cell survival
and growth of myeloma cells, and (3) developing means to enhance host immune
response against myeloma cells. However, no matter what new approach
clinicians may choose to investigate, a thorough understanding of the biology
of myeloma will be necessary for developing effective therapeutic approaches.
Understanding the biological principles that regulate myeloma cell survival
and growth will likely provide new targets for therapy and perhaps improve the
efficacy of already available myeloma treatments.
III. Advances in the Biology and Treatment of Myeloma Bone Disease
James R. Berenson, M.D.*
Biology
The major clinical manifestations of multiple myeloma are related to loss
of bone. This bone loss often leads to pathologic fractures, spinal cord
compression, hypercalcemia and bone pain. As a result, these patients often
require analgesics, radiation therapy and surgery to bone. This enhanced bone
loss occurs because of the stimulation of the cells responsible for bone
resorption, the osteoclasts, and results from the interplay between the tumor
cells, other nonmalignant cells in the bone marrow microenvironment and the
bone-resorbing osteoclasts. Specific soluble factors have been identified in
the bone marrow from myeloma patients that stimulate osteoclasts. Recent
studies have elucidated the specific factors involved in osteoclast
stimulation and the ways that the different cell populations interact in the
bone marrow to produce enhanced bone resorption in these patients.
Bone Resorbing Factors
Initially, Mundy and colleagues identified a number of proteins known as
OAFs, osteoclastic activating factors, which were thought to be the proteins
responsible for enhanced bone loss in myeloma patients. These factors
including lymphotoxin (tumor necrosis factor) (TNF) ß) and interleukin 1
ß (IL-1) ß were identified in the supernatants from cultures from
myeloma cell lines and fresh myeloma bone marrow in these early studies.
However, more recent studies have suggested that other factors are important
in leading to the loss of bone among these patients.
The role of lymphotoxin in myeloma bone disease has been downplayed by more
recent studies failing to find significant differences in the amount of this
cytokine in supernatants derived from bone marrow cultures or fresh bone
marrow plasma derived from myeloma patients compared to controls. In addition,
antibodies to lymphotoxin do not reduce the bone resorbing activity of fresh
bone marrow plasma from myeloma patients. Another tumor necrosis factor,
TNF, is found at higher levels in supernatants from these patients'
bone marrow cultures, and is capable of stimulating osteoclast formation. Its
effects are mediated by stimulation of the proteolytic breakdown of IB
that leads to the release of NFB. This enhancer translocates into the
nucleus where it induces transcription of specific genes, some of which are
involved in enhancing bone resorption. The importance of NFB in bone
resorption is supported by recent studies showing that NFB knockout
mice show osteopetrotic bones.
A recently identified receptor for activation of NFB, a member of
the TNF receptor family, and its ligand, RANKL, has been shown to be key
players in the development of osteoclasts. Unlike other soluble bone resorbing
factors, the activity of these molecules requires direct cell-to-cell contact.
It has been known for some time that osteoclastogenesis requires the direct
interaction of osteoblasts or stromal cells with osteoclasts. The
identification of RANK expressed on the surface of osteoclasts and RANKL on
osteoblasts and stromal cells explains how this interaction leads to
osteoclast development. TNF itself is capable of stimulating osteoblasts to
increase expression of RANKL. Malignant plasma cells from myeloma patients
have been recently shown to express RANKL so that it is possible that the
tumor cells themselves may directly stimulate osteoclast development in the
myeloma bone marrow environment. Importantly, a soluble decoy receptor called
osteoprotegerin (OPG) exists that binds RANKL and prevents the binding of the
ligand to RANK. In fact, animals lacking OPG show profound osteoporosis. It is
the delicate balance between soluble OPG and RANKL that determines the amount
of bone loss. Because of its profound inhibitory effect on bone loss, OPG is
now being evaluated in early clinical trials.
Despite the early findings of Mundy identifying IL-1ß as one of the OAFs in multiple myeloma patients, its role in myeloma bone disease has become less clear. Supernatants from myeloma patients' bone marrow cultures show increased levels of this cytokine.3 The role of IL-1ß in resorbing bone has been shown in bone organ cultures. Although inhibitors of this protein and its receptors are capable of inhibiting bone resorption generated by supernatants derived from myeloma bone marrow, other groups have been unable to show that this cytokine plays a role in myeloma bone disease.2 Fresh bone marrow plasma from myeloma patients does not show higher levels of IL-1ß, and antibodies to this protein do not change the ability of myeloma bone marrow plasma to induce osteoclast formation.
IL-6, which is mainly produced by nonmalignant cells in the bone marrow of myeloma patients, is a cytokine capable of stimulating growth and preventing apoptosis of the malignant cells in myeloma patients. However, it also has been shown to stimulate bone loss by inhibiting bone formation and stimulating osteoclast formation especially in the presence of IL-1 or the soluble IL-6 receptor (sIL-6R). Stromal cells, osteoclasts and osteoblasts are all major sources of IL-6 in the bone marrow microenvironment. Recent studies show that malignant cells from myeloma patients increase IL-6 production by osteoblasts both by direct cell-to-cell contact and release of soluble factors. Since some studies report that malignant cells from myeloma patients produce IL-1 and TNF and both of these cytokines stimulate IL-6 production by osteoblasts,9,10 either or both may be the soluble factor(s) involved in myeloma cell-induced release of IL-6 by osteoblasts. Thus, these studies suggest the role of bone cells not only in bone-related changes in these patients but also in the promotion of growth and prevention of apoptosis of the tumor cells themselves as mediated by IL-6.
IL-11 stimulates osteoclastogenesis and inhibits bone formation.11 It has been shown to be produced by osteoblasts, and present in culture supernatants of bone marrow cells from myeloma patients. This cytokine stimulates RANKL expression by osteoblasts. In addition, recent studies have shown that hepatocyte growth factor (HGF) which is known to be produced by malignant plasma cells,12 may also induce IL-11 secretion by osteoblasts.13 Other cytokines such as IL-1 are capable of potentiating the effect of HGF on IL-11 secretion. High serum levels of HGF are associated with a poor prognosis in myeloma patients.
It is clear that macrophage colony-stimulating factor (M-CSF) is involved in early events in the development of osteoclasts. Although this factor along with RANKL are all that is required for osteoclastogenesis to occur, its role in myeloma bone disease remains unclear. Some studies show higher amounts of this factor in the serum of myeloma patients and its correlation with tumor load.
Recently, macrophage inhibitory protein-1 (MIP-1), has been
identified as an important factor involved in myeloma bone
disease.2 Levels of
this cytokine are elevated in the bone marrow of these patients. This
chemokine is capable of inducing osteoclast formation in vitro, and antibodies
to this protein block the induction of osteoclast formation by fresh bone
marrow plasma from myeloma patients. In addition, this chemokine attracts and
activates monocytes, and is a potent inhibitor of early hematopoiesis.
There is evidence for an increasing role of angiogenesis in the pathogenesis of multiple myeloma. It is clear that vascular endothelial growth factor (VEGF) is produced by malignant plasma cells, and the receptors that bind this factor are expressed on bone marrow stromal cells.14 In fact, recent results show that VEGF increases IL-6 production by bone marrow stromal cells from myeloma patients. This may indirectly lead to enhanced bone loss in these patients. Until recently, it was not clear that VEGF had any direct role in bone resorption. However, it has now been shown that VEGF can replace M-CSF in leading to early osteoclast development.15
Adhesion Molecules
The critical role of the integrin vß3 in
bone resorption has been demonstrated in several recent
studies.16,17
Mice lacking the ß3 molecule show reduced bone resorption, and antibodies
and blocking ligands to vß3 reduce
osteoclastic
activity.16,17
The vß3 molecule binds to a tripeptide, RGD,
within the extracellular matrix. When this integrin is bound to specific
extracellular matrix proteins that contain this peptide, a chain of events
occurs within the osteoclast that results in a cell that is actively resorbing
bone. Both the protein tyrosine kinase Src and another specific kinase, Pyk-2,
are activated upon vß3 binding to the matrix
proteins. The pivotal role of Src in bone resorption is supported by the
development of osteopetrosis in mice without
Src.18 These
animals contain abnormal osteoclasts and lack bone-resorbing activity. In
addition to osteoclasts, vß3 has also been
shown to be present on tumor cells and endothelial
cells.19,20
This molecule is expressed by neovascularized blood vessels and its expression
by tumor cells correlates with invasiveness. Thus, this integrin is important
in bone resorption, angiogenesis, and tumor invasion.
Bone Resorption Markers
A variety of markers of bone resorption and formation have been used to
assess bone disease in myeloma
patients.21,22,23,24
Patients with multiple myeloma show the expected increases in bone resorption
markers such as C-terminal telopeptide of type I collagen, pyridinoline and
deoxypyridinoline and decreases in bone formation markers such as osteocalcin.
In addition, a decrease in osteocalcin level or higher ICTP concentrations
predicts a shortened survival in myeloma. In a recent placebocontrolled
randomized Finnish clinical trial involving oral clodronate, higher baseline
levels of the amino-terminal propeptide of type I procollagen (PINP), a
product of growing osteoblasts, ICTP and alkaline phosphatase (AP) were
associated with a worse survival. PINP and ICTP levels decreased dramatically
during clodronate treatment. Similarly, treatment with oral risedronate
reduced urinary pyridinoline/creatinine and deoxypyridinoline/creatinine
ratios as well as the bone formation markers AP and osteocalcin plasma levels.
Monthly administration of intravenous pamidronate is also associated with a
decrease in both bone resorption and bone formation markers. In the Finnish
clodronate trial, a decrease in these markers during clodronate therapy was
associated with a better survival. In current clinical trials evaluating newer
bisphosphonates, it is being determined whether baseline values or changes in
these markers predict for either the development of new skeletal complications
or whether these agents will be clinically effective in individual cases.
There is some evidence to support this from two recent studies. In breast
cancer patients with lytic bone metastases receiving the aminobisphosphonate
pamidronate who normalized urinary N-telopeptide, a newer bone resorption
marker, levels, there was a reduction in the development of new fractures as
well as progression of bone
metastases.25 In a
recent study evaluating another nitrogen-containing bisphosphonate,
ibandronate, in myeloma patients, increased suppression of urinary bone
resorption markers was associated with reduction in the development of bony
complications.26
Treatment of Myeloma Bone Disease
Although analgesics, surgery and radiotherapy may effectively palliate
patients with complications from myeloma bone disease, these treatments do
little to slow the progressive bone loss that occurs in these patients.
Chemotherapy may reduce tumor burden but has little impact on the underlying
bone disease. The demonstration that bisphosphonates could reduce the skeletal
complications and effectively palliate the symptoms related to bone disease in
myeloma patients has resulted in a dramatic change for the better in the lives
of these patients. Importantly, because these agents lack significant bone
marrow suppressive effects, they can be administered as an adjunct to other
cytotoxic therapy. Recent laboratory studies suggest that these drugs have
potential anti-myeloma effects, and this is supported by clinical studies
showing an improvement in the survival of some patients receiving these drugs.
Newer more potent aminobisphosphonates are in active clinical trials and offer
the potential to further reduce bone-related problems while improving the
overall outcome of myeloma patients. In addition to the bisphosphonates, a
number of other types of anti-bone resorptive agents are entering clinical
trials.
Bisphosphonates
Pharmacology
Bisphosphonates are analogues of endogenous pyrophosphate in which a carbon
atom replaces the central atom of
oxygen.27 This
carbon substitution makes these compounds resistant to hydrolysis, and allows
two additional chains of variable structure. One of these side chains usually
contains a hydroxyl moiety that allows high affinity for calcium crystals and
bone mineral. The differences at the other side chain produce marked
differences in the anti-resorptive potency of different bisphosphonates. In
fact, the newer bisphosphonates, such as ibandronate and zoledronic acid, show
10,000-100,000-fold more potency than do the older agents such as etidronate.
These drugs have limited bioavailability (usually < 1%) and are also poorly
tolerated orally, with significant gastrointestinal toxicity, particularly
esophagitis and esophageal ulcers. The bisphosphonates are almost exclusively
eliminated through renal excretion. Although significant nephrotoxicity can
occur, this is clearly related to the drug dose and rate of intravenous
infusion. Importantly, this renal dysfunction results from the bisphosphonate
backbone so that the newer, more potent bisphosphonates can be administered at
therapeutic doses more rapidly without significant risk of nephrotoxicity.
Bisphosphonates preferentially bind to bones that have high rates of bone turnover (i.e. undergoing increased bone resorption or formation). Thus, these agents are concentrated at the exposed bone surface that undergoes active remodeling. Once these drugs are integrated into a region of bone that is not undergoing remodeling, the bisphosphonates lose their ability to inhibit bone resorption. As a result, continued administration of these agents is required to achieve an enduring reduction in bone resorption in a patient with lytic bone disease.
Mechanisms of action
The inhibition of bone resorption occurs as a result of the effect of these
drugs on osteoclasts both directly and indirectly. Bisphosphonates were first
shown to reduce osteoclast development from their precursors as well as
inhibit movement of osteoclasts to the bone surface where they would normally
resorb bone. These drugs are also capable of inducing apoptosis of osteoclasts
as well as tumor cells from myeloma
patients.28,29
Interestingly, this has been shown to occur as a result of inhibition of the
mevalonic acid pathway particularly in nitrogen-containing
bisphosphonates.29
Interestingly, the statin drugs that lower cholesterol also block enzymes in
this same pathway. Both types of drugs prevent prenylation of a number of
proteins including guanidine triphosphatases such as ras and rho.
Specifically, the addition of geranyl-geranylated derivatives rather than
farnesylated compounds is able to overcome the apoptosis-inducing effects of
aminobisphosphonates and statin
derivatives.29 In
addition, a reduction in the production of the cytokine IL-6 from myeloma bone
marrow stromal cells exposed to bisphosphonates has also been
found.30,31
This cytokine is not only capable of stimulating bone resorption but also is
an important growth factor and anti-apoptotic factor in
myeloma.7 Thus,
reducing the availability of this cytokine in the bone micro-environment by
exposure to bisphosphonates may not only reduce bone loss but may also have an
antimyeloma effect. Recent animal studies have shown that the
aminobisphosphonates also have potent anti-angiogenic activity.
Anti-angigoenesis agents such as thalidomide have recently been shown to be
effective antitumor agents in
myeloma.32 Thus,
the anti-angiogenic effect of the aminobisphosphonates may contribute to these
drugs' antibone resorptive effect (see above) as well as provide additional
mechanisms by which these drugs may have antimyeloma effects as well. A new
potential anti-tumor mechanism for these compounds was recently reported for
aminobisphosphonates.33
These drugs were shown to induce expansion of 
T cells in
peripheral blood mononuclear cell cultures, and enhance cytotoxicity of
malignant plasma cells in bone marrow cultures by these T lymphocytes. Thus,
there is increasing evidence that bisphosphonates, especially the
nitrogen-containing compounds, can lead to direct and indirect effects that
result not only in less bone loss but less tumor burden as well. In support of
this, Epstein and
colleagues34 have
shown a reduction in both lytic bone metastases and improvement in survival in
severe combined immunodeficiency mice implanted with fresh human myeloma bone
marrow and fetal bone after treatment with pamidronate. However, treatment
with ibandronate in a murine model of
myeloma35 showed
only a reduction in lytic bone disease, without an impact on tumor burden.
Clinical studies in myeloma patients
Small, open-label trials involving bisphosphonates suggested their
potential role in these patients. The first two randomized double
blind placebo-controlled trials involved use of daily oral etidronate
(5 mg/kg/d)36 or clodronate
(2,400 mg/d)37 in newly diagnosed patients who
also received oral melphalan and prednisone. Neither of these trials
showed any significant clinical benefit with these less potent oral
agents, although there were fewer patients developing new lytic
lesions in the clodronate trial who received the bisphosphonate. Oral
clodronate has also been the subject of another randomized double
blind trial from the Medical Research Council. 38 In addition to their
chemotherapy, 536 newly diagnosed multiple myeloma patients received
either 1,600 mg of clodronate or placebo daily. Although fewer
patients treated with clodronate experienced severe hypercalcemia and
vertebral as well as nonvertebral fractures, there were no differences
in the time to first skeletal event or requirement for
radiotherapy. The drug had no significant effect on pain except back
pain at a single time point (24 months), and performance status was
also unaffected except at this same time point. Patients receiving
clodronate showed no difference in survival compared to the placebo
group. Oral pamidronate (300 mg/d) was compared to placebo in 300
newly diagnosed myeloma patients also receiving intermittent oral
melphalan and prednisone, and had no effect on skeletal-related
morbidity or survival.39 Several small open-label studies
suggested the possible benefit of infusional pamidronate in myeloma
patients. As a result, 392 myeloma patients with Durie-Salmon Stage
III multiple myeloma and at least one lytic lesion were randomized to
receive monthly 4-hour infusions of either placebo or pamidronate (90
mg) for 21 cycles.40,41
Patients were stratified prior to randomization according to the amount of prior antimyeloma therapy at study entry: stratum 1, first-line chemotherapy; stratum 2, second-line or greater chemotherapy. Initial results after nine cycles showed a marked reduction in the proportion of patients having any skeletal event with pamidronate treatment,40 and this difference was also observed for patients in both stratum 1 and stratum 2. Patients receiving pamidronate also showed decreases in bone pain and no increase in analgesic usage or deterioration in performance status or quality of life, in contrast to placebo patients. The results after an additional 12 cycles of randomized treatment continued to show that the proportion of patients developing any skeletal event remains smaller in the pamidronate group.41 Among all 392 patients, there was no difference in overall survival between the pamidronate and placebo groups. However, patients receiving pamidronate in stratum 2 showed a median survival of 21 months compared with 14 months for placebo patients. Thus, it is clear from this large randomized trial that intravenous pamidronate (90 mg), when administered monthly as an adjunct to chemotherapy, results in a significant reduction in the skeletal complications as well as palliation of symptoms among myeloma patients with lytic bone disease. It is unclear whether this drug or other bisphosphonates will be similarly effective for patients without lytic bone disease or prevent patients with solitary plasmacytoma from developing multiple myeloma. Third-generation bisphosphonates (e.g., zoledronic acid and ibandronate), that appear to be more than 100 times more potent than second-generation aminobisphosphonates, have recently entered clinical trials. Very small doses of these agents effectively restore normocalcemia in patients with tumor-induced hypercalcemia.42,43 Recent results show the superiority of zoledronic acid (4 or 8 mg) compared to pamidronate (90 mg) in reversing hypercalcemia of malignancy.44 Clinical evaluation of zoledronic acid and ibandronate in bone metastases is in progress. Zoledronic acid can be given safely over several minutes and produces similar anti-resorptive effects, as assessed by bone resorption marker, as 90 mg of pamidronate.45 Preliminary reports from a randomized phase II study comparing monthly infusions of zoledronic acid (0.4, 2 or 4 mg as a 5-minute infusion) compared to pamidronate (90 mg as a 2-hour infusion) in 280 patients with lytic bone metastases (109 myeloma) have been reported.46 The proportion of patients with any SRE was lower (30-35%) in the 2-mg and 4-mg zoledronic acid and the pamidronate groups compared to the 0.4 mg zoledronic acid group (46%). This phase II trial was not "powered" to show superiority of zoledronic acid compared to pamidronate. An ongoing, larger phase III randomized trial compares higher doses of zoledronic acid to 90-mg pamidronate in multiple myeloma or breast cancer with lytic disease. Ibandronate is another newer potent bisphosphonate. A phase III placebo-controlled trial of 214 myeloma patients with Durie Salmon Stage II or III was recently completed.26 Patients either received monthly bolus injections of 2 mg of ibandronate or placebo injections in addition to their antineoplastic therapy. Ninety-nine patients in each group were evaluable for efficacy. The mean number of events per patient year on treatment was similar in both groups (ibandronate 2.13 vs placebo 2.05). However, in the subgroup of ibandronate-treated patients showing a sustained reduction in bone resorption markers, less SREs/year occurred. There was no difference in overall survival. Thus, this dose of ibandronate is inadequate to show significant effects on preventing skeletal complications in myeloma.
New Agents
A number of new agents are entering clinical trials for the treatment of
metastatic bone disease. An oral vß3
antagonist, the RGD peptidomimetic SD-7784, is entering a phase I trial in
myeloma patients. vß3 antagonists have
previously been shown to have potent anti-tumor and anti-osteoclastic effects
in animal
models.16,17,20
Thus, these drugs have the potential to both reduce bone loss as well as
reduce tumor burden in myeloma patients. An OPG analog, an inhibitor of
RANK-RANKL interaction, is now being evaluated in a clinical trial in patients
with bone metastases. In addition, inhibitors of src activity show marked
anti-resorptive capability in animal
models47 and may
enter clinical trials soon. The cholesterol-lowering statin drugs have
recently shown to increase bone formation in vitro and in vivo in rodent
models,48 possibly
by their induction of bone morphogenetic protein (BMP)-2 as well as their
ability to induce apoptosis of osteoclasts. Some studies have suggested that
patients receiving statins to lower serum cholesterol also show a reduced risk
of developing fractures. The apoptotic-inducing effect of statins has also
been shown to occur in myeloma cells in vitro through their blockage of the
mevalonic acid
pathway.29 Whether
either the bone-enhancing or anti-tumor effects of statins is clinically
observed in myeloma patients is being assessed in clinical trials. New
attempts are underway to encourage new bone formation using BMPs, gene therapy
and mesenchymal stem
cells.49 Thus, the
potential exists to not only halt the relentless bone loss in myeloma patients
but to repair their already damaged bones.
IV. Novel Biologically Based Therapies for Myeloma
Kenneth C. Anderson, M.D.*
Novel Therapeutic Targets
Multiple myeloma is incurable with current therapies, but several recent
biological advances have provided the framework for novel therapeutic
strategies. First, multiple lines of evidence suggest that the precursor cell
in multiple myeloma is a cytoplasmic µ-positive B cell that has undergone
antigen selection and somatic hypermutation in the lymph node, but which has
not yet undergone isotype class switching. Chromosomal translocations
involving the immunoglobulin (Ig) switch region are common, and multiple
partner chromosomes have been described. Given that abnormalities in Ig gene
rearrangement, IgH class switching, and DNA damage repair are hallmarks of
myeloma, we have undertaken studies of Ku expression and function in human
myeloma cells.1 Ku
is a heterodimer composed of Ku70 and Ku86 subunits that binds with high
affinity to altered DNA and is essential for double stranded DNA break (DSB)
repair and normal Ig V(D)J recombination. Our studies to date have identified
a 69 kD variant of Ku86 (Ku86v) in some myeloma cells, which neither binds
DNA-dependent protein kinase (DNA-PKcs) nor activates kinase activity and
therefore may account for decreased DNA repair and increased sensitivity to
radiation and chemotherapy; conversely, Ku86 in myeloma cells confers
resistance to therapy and may represent a therapeutic target.
Myeloma cells home to the bone marrow (BM) microenvironment, where excess
plasma cells characteristic of this disease accumulate. We have demonstrated
mechanisms whereby tumor cells specifically adhere both to extracellular
matrix (ECM) proteins and bone marrow stromal cells (BMSCs), as well as
changes in cell adhesion molecule profile correlating with egress of tumor
cells into the peripheral blood (PB) in the context of progressive disease and
plasma cell leukemia
(PCL).2 Adhesion
molecules not only localize tumor cells within the BM microenvironment but
also have multiple functional sequelae. Adherence to BMSCs confers resistance
to apoptosis,3 and
agents that block adhesion, i.e. bisphosphonates, can confer sensitivity to
treatment. Adherence of tumor cells to BMSCs upregulates IL-6 transcription
dependent in part on the transcription factor NFkB, as well as IL-6 secretion
within BMSCs,4 and
also allows for tumor cell secretion of cytokines, i.e., transforming growth
factor ß, which further enhances IL-6 transcription and secretion in
BMSCs.5 This is of
central importance since our studies have shown that IL-6 is both a growth and
survival factor for myeloma
cells.6 Proteasome
inhibitors are novel drugs that inhibit activation of
NFB7; they
induce apoptosis of myeloma cells which are resistant to conventional therapy
and importantly, inhibit the NFB-dependent upregulation of IL-6 in
BMSCs and related paracrine growth of adherent tumor
cells.8
We have shown that proliferation of myeloma cells triggered by IL-6 is mediated via the mitogen-activated protein kinase (MAPK) cascade,9 suggesting therapeutic strategies based upon blocking this pathway in tumor cells. Apoptosis triggered by gamma irradiation (IR), Fas, and Dexamethasone (Dex) is mediated via distinct signaling cascades. For example, Dex (but not IR or Fas)-induced apoptosis is mediated via activation of related adhesion focal tyrosine kinase (RAFTK).10 IL-6 is also a survival factor for human myeloma cells, specifically activating protein tyrosine phosphatase (SHP2) and thereby blocking the activation of RAFTK and related apoptosis in response to Dex.11 Blocking SHP2 activation with small molecule SHP2 inhibitors may therefore relieve this protective effect. Further delineation of these pathways will derive strategies for triggering apoptosis, overcoming Dex resistance, and inhibiting survival signals, which will provide the framework for related novel treatment approaches.12
Our recent studies also suggest that adhesion of myeloma cells to BMSCs
also upregulates vascular endothelial growth factor (VEGF) secretion by BMSCs
and myeloma cells. Therefore, in addition to examining the effect of VEGF on
BM angiogenesis, we are evaluating whether VEGF is a growth and/or survival
factor for myeloma cells. Preliminary studies suggest that VEGF induces MAPK
activation and proliferation of some myeloma cells, and that VEGF receptor
inhibitors block proliferation of tumor cells and may therefore be useful
clinically. This increase in VEGF may in part account for increased
angiogenesis in human myeloma BM. Based upon its anti-angiogenic activity,
thalidomide (Thal) was recently used very successfully to treat patients with
myeloma, even those refractory to conventional
therapy.13
Although Thal may be acting in myeloma as an anti-angiogenic agent, there are
multiple other potential mechanisms of action of Thal and/or its in vivo
metabolites.14
First, Thal may have a direct effect on the myeloma cell and/or BMSC cell to
inhibit growth and survival. For example, free radical-mediated oxidative DNA
damage may play a role in the teratogenicity of Thal and may also have
anti-tumor effects. Second, adhesion of myeloma cells to BMSCs both triggers
secretion of cytokines that augment myeloma cell growth and survival and
confers drug resistance; thalidomide modulates adhesive interactions and
thereby may alter tumor cell growth, survival, and drug resistance. Third,
cytokines secreted into the BM microenvironment by myeloma and/or BMSCs, such
as IL-6, IL-1ß, IL-10 and TNF, may augment myeloma cell growth and
survival, and Thal may alter their secretion and bioactivity. Fourth, VEGF and
basic fibroblast growth factor (bFGF)-2 are secreted by myeloma and/or BMSCs
and may play a role both in tumor cell growth and survival, as well as BM
angiogenesis. Given its known anti-angiogenic activity, Thal may inhibit
activity of VEGF, bFGF-2, and/or angiogenesis in myeloma. Finally, Thal may be
acting against myeloma via its immunomodulatory effects, such as induction of
a Th1 T cell response with secretion of interferon gamma (IFN-) and
IL-2. Understanding which of these mechanisms mediate anti-myeloma activity
will be critical both to optimally define its clinical utility and to derive
analogues with enhanced potency and fewer side effects. Already two classes of
Thal analogues have been reported, including phosphodiesterase 4 inhibitors
that inhibit TNF but have little effect on T cell activation, and
others that are not phosphodiesterase inhibitors but do markedly stimulate T
cell proliferation as well as IFN- and IL-2
secretion.15 In
recent studies, we have delineated mechanisms of anti-tumor activity of Thal
and its potent analogoues (immunomodulatory drugs,
IMiDs).16
Importantly, these agents act directly, via inducing apoptosis or G1 growth
arrest, in myeloma cell lines and patient myeloma cells that are resistant to
melphalan, doxorubicin, and Dex. Moreover, Thal and the IMiDs enhance the
anti-myeloma activity of Dex and, similar to Dex, apoptotic signaling
triggered by Thal and the IMiDs is associated with activation of RAFTK. Most
recent studies suggest that treatment with these drugs augments their
adherence to BMSCs and fibronectin, but abrogates the upregulation of IL-6 and
VEGF induced by tumor cell binding. Finally, these drugs appear to upregulate
natural killer cell-mediated killing of myeloma cells. These studies establish
the framework for the development and testing of Thal and the IMiDs in a new
treatment paradigm to target both the tumor cell and the microenvironment,
overcome classical drug resistance, and achieve improved outcome in this
presently incurable disease.
Immune-Based Strategies
Although high response rates can be achieved using high-dose therapy
followed by stem cell grafting, the majority of patients are destined to
relapse and few, if any, are cured. Major obstacles to cure are the excessive
toxicity noted after allografting in myeloma, contaminating tumor cells in
autografts, and most importantly, the persistence of minimal residual disease
(MRD) after high-dose therapy followed by either allogeneic or autologous stem
cell transplantation. In this context, we are developing improved strategies
to treat MRD after high-dose therapy followed by allogeneic or autologous stem
cell grafting. Most importantly, we are developing multiple approaches for the
generation and enhancement of allogeneic and autologous anti-myeloma immunity
in vitro and in animal models. Based upon these studies, we are designing
clinical trials that couple our treatments to achieve MRD with these novel
immune-based therapies post transplant in an attempt to achieve long-term
disease-free survival and potential cure of multiple myeloma.
Allografting
We have carried out high dose therapy followed by T (CD6) cell-depleted
allografting using histocompatible sibling donors in 61 patients with myeloma
whose disease remained sensitive to conventional
chemotherapy.17
This included 39 men and 22 women with median age of 44 (32-55) years. Most
patients presented with advanced stage myeloma. The majority of patients
achieved either complete (28%) or partial (57%) response; importantly, only
17% patients developed grade 2 graft-versus-host disease (GVHD), and the
transplant-related mortality was only 5%. Therefore we have shown that
allografting can be done safely in myeloma. Indeed in our Center the over-all
and progression-free survival of allograft and autograft recipients is
equivalent, with approximately 40% patients surviving at 3 years. However,
only 20% patients are disease free at 4 years posttransplant. Excitingly,
data from our centers and others unequivocally demonstrate that donor
lymphocyte infusions (DLI) mediate a graft-versus-myeloma effect (GVM) which
can effectively treat relapsed myeloma post
allografting.18,19
Unfortunately GVHD is a frequent cause of morbidity and mortality after DLI.
However, at our Myeloma Center five of seven patients who had relapsed
post-CD6-depleted allografting responded (including three complete responses)
to CD4+ T cell enriched DLI, in some cases in the absence of GVHD. This raised
the possibility that distinct T cell clones may be mediating GVM versus GVHD.
Given the high response rates but inevitable relapses observed in the setting
of allografting for myeloma, we are now testing in a clinical protocol whether
CD4+ DLI at 6 months post-CD6-depleted allografting may mediate GVM, which
will effectively treat MRD and thereby improve outcome. To date 21 patients
have undergone CD6-depleted allografting, 18 of whom developed only grades 0-1
GVHD. Eleven of these 18 patients are > 6 months posttransplant and have
received CD4+ DLI. Eight of the 11 patients who received DLI demonstrated
further response (including four complete responses), suggesting the potential
of DLI to treat MRD. Therefore our studies already suggest that GVM can be
adoptively transferred in this fashion. We are also examining T cell
repertoire, based upon Vß T cell receptor gene rearrangement, to identify
those clonal T cells associated with GVM and their target antigens on tumor
cells.20,21
Already we have shown that T cells mediating GVM can target idiotypic antigens
and are presently identifying other target antigens. The goal of these studies
is to characterize, isolate, and expand GVMT cell clones for antigen-specific
adoptive immunotherapy.
Autografting
Although randomized studies convincingly demonstrate a survival advantage
for myeloma patients treated with high-dose therapy and autografting compared
to those receiving conventional chemotherapy, this treatment is not curative.
Two sites of MRD contribute to the failure of autografting: MRD in the
autograft and MRD in the patient post myeloablative therapy. At our Center we
have to date carried out high dose therapy and stem cell autografting in 105
patients who presented with advanced stage myeloma but whose disease remained
sensitive to chemotherapy. As in our allografting experience, the majority of
patients responded, including 30% complete and 62% partial responses. However,
none of these patients are cured. We have produced monoclonal antibodies in
the laboratory that have been used to deplete tumor cells from myeloma
autografts.22 We
have also evaluated CD34 selection techniques to select normal hematopoietic
progenitor cells within
autografts.23
However, these methods deplete only 2-3 logs of tumor cells, and > 50%
autografts still contain MRD. Based upon our laboratory data that myeloma
cells express Muc-1 and adenoviral receptors, we have specifically transduced
tumor cells within myeloma autografts with the thymidine kinase (tK) gene
using an adenoviral vector with a tumor selective (Muc-1) promotor, followed
by purging tumor cells exvivo by treatment with
ganciclovir.24
Pilot studies suggest that > 6-7 logs of tumor cells can be purged under
conditions that do not adversely affect normal hematopoietic progenitor cells,
setting the stage for a clinical trial of adenoviral purging prior to
autotransplantation.
We are also attempting to generate and expand anti-myeloma specific autologous T cells ex vivo for adoptive immunotherapy of MRD in the patient post autotransplant. It is now possible to clone the gene for the patient's specific idiotypic protein, use computer programs to identify gene sequences encoding for peptides predicted to be presented within the groove of Class I HLA of a given patient's HLA type, and expand peptide specific T cells ex vivo.25 A similar strategy can be used to expand T cells against peptides within shared antigens that are overexpressed on myeloma cells, such as telomerase catalytic subunit (hTERT),26 Muc-1,27 or CYP1B1.28 Strategies are also being tested to enhance the immunogenicity of the whole tumor cell. Our laboratory studies have shown that autologous T cells do not proliferate to the patients' own tumor cells as targets in an autologous MLR. However, CD40 activation of myeloma cells upregulates Class I and II HLA, costimulatory, GRP94, and other molecules; and CD40 activated myeloma cells trigger a brisk autologous T cell response.29 T cells can therefore be harvested from myeloma patients before autografting, expanded ex vivo using CD40-activated autologous myeloma cells as stimuli, and given as adoptive immunotherapy to treat MRD post transplant. Finally, we are developing and examining the clinical utility of a variety of myeloma vaccines. First, based upon our observation that CD40-activated myeloma cells trigger a brisk autologous T cell response, we will examine the utility of vaccinations of patients with autologous CD40 activated tumor cells. Second, based upon our demonstration of the expression of Muc-1 core protein on freshly isolated myeloma cells,27 we will construct and evaluate two vaccines: recombinant vaccinia virus containing the Muc-1 gene and autologous dendritic cells (DCs) transduced using adenoviral vectors with Muc-1. Excitingly, we have recently shown that myeloma cells can be fused to DCs and that the use of the myeloma cell-DC fusion as an antigen presenting cell presents the entire myeloma cell as foreign. In a syngeneic murine myeloma model, vaccinations with myeloma cell-DC fusions, but not with either myeloma cells or DCs alone, demonstrated both protective and therapeutic efficacy. Most importantly, we have shown that patient myeloma cells can be fused to autologous DCs, which are readily isolated from either patient bone marrow and peripheral blood,30 and that autologous myeloma cell-DC fusions can trigger specific cytolytic autologous T cell responses in vitro.31 We will therefore translate these findings to the bedside in clinical trials of myeloma-DC fusion vaccines to assess in vivo myeloma-specific T and B cell responses as well as clinical efficacy. Ultimately, vaccinations will be coupled with adoptive immunotherapy in an attempt to treat MRD post autografting and thereby improve outcome.
Footnotes
* Mayo Clinic, Room 920 Hilton Bldg., 200 First Street SW, Rochester MN
55905 
* Clinical Investigations, H. Lee Moffitt Cancer Center, 12902 Magnolia
Drive, MRC 3043, Tampa FL 33612-9497
* Department of Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd.,
Bev. Mod.-1, Room 120, Los Angeles CA 90048
* Dana-Farber Cancer Institute, 44 Binney Street, Boston MA 02115
I. Current Therapy of Myeloma
therapy. Bone Marrow Transplant. 1999; 24: 13
-17.[CrossRef][Medline]
