Chapter 8
Summary, general discussion and
future perspectives
Summary
Multiple Myeloma (MM) is characterized by a malignant proliferation of monoclonal plasma cells in the bone marrow, clinical presenting by skeletal destruction, renal failure, anemia and hypercalcemia. MM accounts for 10% of all hematological malignancies and can be preceded by premalignant MGUS. In the Netherlands the annual incidence is 850 cases and increases progressively with age.
In chapter 1 the normal B-cell differentiation and tumor development of B-cells are described, including the development of malignant plasma cells in multiple myeloma. The Durie–Salmon staging system is the most commonly used staging system for patients with multiple myeloma. Recently, an
International Staging System for MM has been proposed. This staging system divides patients into three stages based simply on serum β2-microglobulin and albumin levels by which median survivals can be predicted. A number of additional prognostic factors, pertinent to standard dose therapy, have also shown to be relevant with high-dose chemotherapy. These include CRP, plasma cell labeling index, LDH, cytogenetics and surface markers which are also described in the introduction. The treatment of MM during the last three decades has consisted of alkylating agents with prednisolone.
Long-term survivors are unusual with this treatment modality and the median survival duration is 24-36 months. Further increase in dose intensity can be obtained by performing autologous stem cell transplantation. Today intensive chemotherapy with high dose melphalan with autologous stem cell support is the standard therapy for multiple myeloma patients younger than 65 years in the Netherlands. Despite longer median EFS and OS with this intensive treatment, the disease is still incurable in most of the cases.
Further advances in treatment can be obtained from studying cellular processes and apoptosis in MM. Apoptosis is induced through two distinct pathways: (1) the extrinsic or death receptor pathway composed of tumor necrosis factor-family receptors and ligands and (2) the intrinsic pathway with the release of mitochondrial constituents. The possibility of inducing apoptosis will require that great care has to be taken in the initial evaluation of new agents. Drugs that simultaneously target anti-apoptotic proteins, while lethal to tumor cells, are likely to be very toxic to the host. Specific agents are therefore
Thalidomide exerts its antimyeloma effect through multiple mechanisms including anti-angiogenesis, immunomodulation and induction of apoptosis in tumor cells, as well as its effect on the tumor microenvironment.
Bortezomib (formerly PS-341) is a small molecule that is a potent and selective inhibitor of the 26S proteasome which is the primary component of the protein degradation pathway of the cell. Bortezomib inhibits proliferation and induces apoptosis of human myeloma cell lines.
It also inhibits nuclear factor-kappa B (NF-κB) activation, overcomes drug resistance and adds to the anti-myeloma activity of dexamethasone in vitro. The first autologous stem transplants were performed in the 80-ties. In the ongoing years this transplantation option was introduced in the
Netherlands and the first results of patients treated in Groningen are described in chapter 2. The applied regimen consisted of VAD induction chemotherapy. In the case of responsive disease, leukapheresis was performed after high-dose cyclophosphamide followed by G-CSF.
After normalization of peripheral blood counts patients were treated by etoposide, cisplatinum, dexamethasone and ara-C (EDAP). Finally, the treatment was followed by high-dose melphalan in combination with peripheral blood stem cell infusion. EDAP was chosen since its lack of cross resistance. By using two forms of G-CSF with different doses,
high numbers of CD34+ cells were harvested in most cases, indicating that
a much lower dose of G-CSF could be applied. After the total course the overall response was 84% with a complete remission rate of 30%.
The median event-free and overall survival was 29.0 and 44.0 months respectively with a median follow-up of 33 months. Post-transplantation a high incidence of oligoclonal serum Igs was observed in 73% of the patients. We demonstrated by ASO-PCR and sequencing techniques that the occurrence of oligoclonal serum Igs post transplantation was not caused by myeloma related clonal B-cells but rather by the regenerating B-cell compartment. These results indicate that the oligoclonal serum Igs post transplantation cannot be considered as a sign of tumor relapse.
In chapter 3 the same treatment regimen is described for 3 patients with plasma cell Leukemia (PCL) together with a review of the literature.
PCL, the leukemia variant of malignant plasma cell disorders, is a rare disease and is the least common variant of multiple myeloma accounting for 2-3% of all plasma cell dyscrasias.
The primary form occurs in individuals without preceding MM and normally with a rapid clinical course and a short survival. Although just three patients were described, the long remission time of two patients suggest that intensive chemotherapy for PCL might be a promising approach.
Phase I-II studies have demonstrated that thalidomide at a dose of 200-800 mg is an effective agent for patients with refractory MM, with a 50% M-protein reduction in one-third of the patients. However, this dose is associated with a distinct toxicity profile. Lower doses of thalidomide have also been studied mostly in combination with dexamethasone resulting in a response rate of around 55%. This combination has the drawback of an increased risk of thrombosis in 10% of the patients, necessitating the use of prophylactic anticoagulation.
In chapter 4 we studied whether lower doses of thalidomide in combination with daily dose of cyclophosphamide might be an effective regimen with fewer side effects. Low-doses of continuous cyclophosphamide, also called
metronomic scheduling, minimizes toxic side-effects and eliminates the obligatory rest periods between cycles, resulting in the continuous suppression of the malignant clone. In addition to enhancing the direct cytotoxic effect on the myeloma cells, combining thalidomide with cyclophosphamide could also target the inhibition of angiogenesis in the bone marrow microenvironment. We included 38 patients with relapsed or refractory MM. Side-effects were observed in all patients, with neurotoxicity as the most troublesome. With a median follow-up of 12 months 84% of the patients responded, including 64% partial response. The median time of progression free survival was 30 months and the median overall survival time was 20 months.
Myeloma cells are exposed to multiple noxious stimuli induced by different forms of therapy. Resistance to apoptosis in these cases ultimately depends on the ability of the MM cells to prevent activation of the mitochondrial pathway of apoptosis. Whether noxious signals are able to activate this pathway is determined by members of the anti-apoptotic Bcl-2 family. Understanding the molecular functions of these proteins is required for the design of novel
therapeutics to overcome resistance to apoptosis.
Anti-apoptotic family members (Bcl-2, Bcl-xl, Mcl-1) are localized to the outer mitochondrial membrane of multiple myeloma cells.
In chapter 5 the immunohistochemical expression of Bcl-xl, a downstream target of the IL-6 controlled STAT-3 was studied in 40 multiple myeloma bone
marrow samples before treatment and in 11 multiple myeloma bone marrow samples at relapse. The results were compared with the Bcl-xl expression in normal bone marrow plasma cells. The results indicated that Bcl-xl is elevated in a subgroup of MM patients which so far did not correlate with clinical outcome.
MM plasma cells can be distinguished from normal plasma cells on basis of the expression of several surface markers. One of this surface marker might be CD27. CD27 is a transmembrane glycoprotein of the Tumor Necrosis Factor Receptor family. CD27 is expressed by a subset of B-cells and by the majority of peripheral T-cells. The natural ligand for CD27 is CD70,
which is expressed on activated B- and T-cells. The CD27/CD70 interaction is implicated in B-cell differentiation and survival. In two previous studies it has been suggested that CD27 is not expressed on MM plasma cells.
In chapter 6 a comprehensive analysis of CD27 expression in a cross-sectional MM patient group was studied. CD27 was variable expressed on malignant plasma cells. Importantly, plasma cells from MM patients in complete clinical remission display a significant higher CD27 expression compared to plasma cells obtained from newly diagnosed, relapsed and refractory MM patients. In a previous study it was shown that gene profiling of newly diagnosed MM patients identified hierarchical clustered MM subgroups, which are associated with the prevalence of established adverse prognostic factors. In this study we demonstrate that CD27 is differentially expressed in hierarchical clustered MM subgroups, by reanalysis of the previously published cDNA microarray data set. MM patients displaying the lowest CD27 expression were all clustered in the high-risk patients groups, of which the mRNA expression profile closely resembles that of human MM cell lines.
As a result of this study the expression of CD27 on malignant plasma cells was studied on CD38+/CD138+ plasma cells from 67 multiple myeloma (MM)
patients upfront treatment. The results are discussed in chapter 7. No prognostic value of CD27 expression level could be found.
General discussion and future perspectives
Plasma cells are terminally differentiated cells of the B-lymphocyte lineage. They are thought of as cellular factories whose entire energy and synthetic capacity is devoted to producing single antibody protein.
They are normally incapable of dividing and are thought to have a relative short life span of perhaps several weeks. Plasma cells are abundant both in the lymph nodes, where they are found predominantly in the medullar cords and in the marrow. They have distinctive morphology and are easily identified in the marrow.
The most common malignancy involving plasma cells is multiple myeloma (MM). It is an interesting and instructive disease from the standpoint of diagnosis and pathophysiology. However, from the standpoint of therapy it is one of the most challenging disorders of the hematological malignancies. In chapter 2 and 4 two clinical trials are described.
The clinical trial in chapter 2 was a phase II clinical study.
At that time we had modified the protocol by adding an extra course of EDAP chemotherapy after the CAD mobilization chemotherapy (EDAP protocol).191
The rationale for this additional chemotherapy was to circumvent cross resistance for VAD.148
Recently, we have updated the treatment results (EFS and OS) of this EDAP protocol and compared the results of patients treated according to the HOVON 24 (n=28) and HOVON 50 (n=40) protocol (see figure 8.1). In the HOVON 24 protocol the efficacy of intensified chemotherapy (melphalan 140 mg/m2 divided in 2 cycles) followed by myelo-ablative
chemotherapy (cyclophosphamide 60 mg/kg and total body irradiation) and ASCT was compared with intensified chemotherapy alone in newly diagnosed MM patients. In the HOVON 50 protocol the effect of thalidomide combined with Adriamycin, Dexamethasone (TAD) and HDM followed by thalidomide maintenance therapy was studied in patients with multiple myeloma.
The patients characteristics and staging according to Durie and salmon staging system was comparable for all three groups.
Post-transplantation, the median follow-up of these studies were 40 (0-142), 43 (3-94) and 25 months (1-41), respectively. No significant difference in EFS and OS was observed between these three treatment regimens. Figure 8.1a and figure 8.1b present the EFS and OS of the patients since transplantation. We also analyzed the EFS and OS from time of diagnosis as depicted in figures 8.1c and figure 8.1d.
Figure 8.1c demonstrates that the EFS of the EDAP protocol was significantly better than the EFS of the HOVON 50 protocol (P=0.042).
Figure 8.1 Kaplan-Meier estimates of distributions of Event Free Survival (EFS) and Overall Survival (OS) based on patients with multiple myeloma treated according to the EDAP, HOVON 24 and HOVON 50 protocol. A and B demonstrate the EFS and OS since transplantation;
C and D demonstrate EFS and OS since diagnosis.
the EDAP protocol was not inferior in terms of EFS and OS.
These findings suggest that the EDAP protocol might be applied as first-line treatment in MM patients before transplantation.
Although MM still remains an incurable disease, major advances have occurred in our understanding of the biology of MM and in its treatment in the past decade. New diagnostic criteria have been developed and an
international Staging System has replaced the Durie-Salmon Staging System.6
It is now possible to classify MM as standard risk or high risk on the basis of specific independent prognostic factors.
125 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 125 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 125 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 125 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=36) HOVON 24 (n=28) HOVON 50 (n=30) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) 0 25 50 75 100 125 150 175 200 0 20 40 60 80 100 EDAP (n=37) HOVON 24 (n=28) HOVON 50 (n=44) A B D C EFS (%) EFS (%) Ov erall S ur viv al (%) Ov erall S ur viv al (%)
Time (months) Time (months)
Time (months) Time (months)
EFS since Tx OS since Tx
The role of single and double ASCT has been clarified by randomized trials.113,115 Most importantly, thalidomide, bortezomib
and lenalidomide have emerged as new active agents and are being incorporated rapidly into the treatment of both newly diagnosed and relapsed MM.
The Inter Groupe Francophonedu Myélome reported seven-year event-free and overallsurvival rates of 20 and 40 percent, respectively — twicethe rates obtained with single transplantations.115,176,255
The addition of thalidomide to intensive melphalan-based chemotherapy supported with two peripheral-blood hematopoietic stem-celltransplantations improved the rate of complete response andEFS among patients with newly diagnosed multiplemyeloma. The drug failed, however, to prolong OSand was associated with considerable adverse effects.256 Attalet al. conducteda
randomized trial of maintenance treatment with thalidomideand pamidronate. Two months after high-dose therapy, 597 patientsyounger than the age of 65 years were randomly assigned to receiveno maintenance (arm A), pamidronate (arm B), or pamidronateplus thalidomide (arm C). A complete or very good partial responsewas achieved by 55% of patients in arm A, 57% in arm B and 67% in arm C (P=0.03). The 3-year post randomization probabilityof EFS was 36% in arm A, 37% in arm B and 52%in arm C (P<0.009). The 4 year
post-diagnosis probabilityof survival was 77% in arm A, 74% in arm B and 87% in arm C(P<0.04). The proportion of patients who had skeletal eventswas 24% in arm A, 21% in arm B and 18% in arm C (P=0.4). In conclusion, these studies demonstrate that thalidomideis an effective maintenance therapy in patients with multiplemyeloma. However, maintenance treatment with pamidronate does not decreasethe incidence of bone events.257
In conclusion, these results demonstrate that thalidomide increased response rates with prolongation of the EFS.
However, the significance for OS is not well defined.256,257
Superior response rates have been reported for thalidomide plusdexamethasone as compared with dexamethasone alone for inductiontherapy in patients with multiple myeloma.86,88 Since many patientsin these trials received high-dose
therapy after induction therapywith thalidomide and dexamethasone, the long-term benefit ofthis combination cannot be ascertained.
The trombotic risk is highest during induction therapy,when the burden of tumor is high.258 The incidence of neurotoxiceffects should be reduced with the
maintenance therapy. Highrates of complete response approaching the rates observed withhigh-dose chemotherapy plus stem-cell transplantation have recently been described in trials of thalidomide combined with standard treatment with melphalan and prednisone.259
Similarly, combinationsof bortezomib and dexamethasone,96,260 plus pegylated
doxorubicin261 or thalidomide,109,262 have shown promise. Little is known,
however,about the durability of the responses induced by these treatments, especially after the discontinuation of the drugs. Althoughstandard high-dose melphalan therapy with stem-cell transplantationhas considerable acute adverse effects, its low mortality andinfrequent chronic adverse effects have to be balanced againstthe potential of the newer agents for irreversible and incapacitatingchronic adverse effects. Acute complications
(thromboembolismwith thalidomide and lenalidomide) and chronic sequelae (polyneuropathywith thalidomide and bortezomib) may be minimized by combining“old” and “new” therapies, especially since the genomic
heterogeneityof multiple myeloma may require a multifaceted approach to treatmentto achieve lasting control.263 Anticoagulant prophylaxis reduces the
risk of thromboembolism.264 Daily low-dose aspirin (81 mg orally) given to
patients with newly diagnosed and relapsed/refractory multiple myeloma who were receiving pegylated doxorubicin, vincristine and decreased-frequency dexamethasone, plus thalidomide (DVd-T) reduced the incidence of venous thromboembolism without an increase in bleeding complications.265
Protein tyrosine kinases (TKs) are enzymes that catalyze thetransfer of
phosphate from ATP to tyrosine residues in polypeptides. The human genome contains about 90 TK and 43 TK-like genes,the products of which regulate cellular proliferation, survival,differentiation, function and motility.
More than 25 yearsago, TKs were implicated as oncogenes in animal tumors inducedby retroviruses. However, they were largely ignored in drug
development because of a paucity of evidence for a causativerole in human cancer and concerns about drug specificity andtoxicity. The landscape was changed radically by the successof imatinib mesylate, an inhibitor of the BCR-ABL TK in chronicmyeloid leukemia (CML) - a result heralded as a proof-of-principleand a triumph of targeted cancer therapy.
TKs are now regardedas excellent targets for cancer chemotherapy, but reality liessomewhere between the extremes of triumph and tribulation.266
these diseases are in their infancy. Fusionof the fibroblast growth factor receptor 1 (FGFR1) TK with oneof several partners occurs in the 8p11 myeloproliferative syndrome and FGFR3 is mutated and overexpressed in multiple myeloma witht(4;14).267
A recent study of Trudel et al demonstrated that PRO-001, a highly specific anti-FGFR3-neutralizing antibody is a potent and specific inhibitor of FGFR3 and deserves further study for the treatment of FGFR3-expressing myeloma.268
Myeloma cells grow in the bone marrow, where the microenvironment supportstheir growth and protects them from apoptosis. The accumulationof myeloma cells within the bone marrow is associated with increasedrates of bone turnover. RANKL and OPG play an essential role in osteoclast formation and activation. The interaction of MM with stroma resultsin deregulation of the RANKL/OPG axis, both in increasing RANKLand decreasing OPG. This disruption of the RANKL/OPG ratio inthe bone environment increases osteoclast activity, triggersbone destruction and promotes tumor growth. Osteoclast inhibitors(bisphosphonates or specific inhibitors of RANKL) halted MM-inducedbone resorption and resulted in inhibition of myeloma growth and survival. In line with this concept, a recent study has shown that IL-6and osteopontin highly produced by osteoclasts played a centralrole in survival and growth of myeloma cells.269
Indeed,the use of effective osteoclast inhibitors in vivo could breakdown this vicious circle and both suppress bone resorption anddecrease tumor growth. It is tempting to speculate that interferingwith bone marrow cultivation by myeloma cells may inhibit the developmentof myeloma especially in early or premalignant stages (MGUS).Altogether, these observations strongly suggest that reducingthe RANKL/OPG ratio by treatment with RANKL inhibitors and/orMIP inhibitors should provide a high therapeutic interest todecrease both bone resorption and tumor burden in MM.
Bisphosphonates are effective in the prevention and treatment of bone disease in multiple myeloma. Osteonecrosis of the jaw is increasingly recognized as a serious complication of long-term bisphosphonate therapy.
Intravenous pamidronate and intravenous zoledronic acid are equally effective and superior to placebo in reducing skeletal complications.
Pamidronate is favored over zoledronic acid until more data are available on the risk of complications, i.e. osteonecrosis of the jaw. Lacy et al et al recommend discontinuing bisphosphonates after 2 years of therapy for patients who achieve
complete response and/or plateau phase. For patients whose disease is active, who have not achieved a response, or who have threatening bone disease beyond 2 years, therapy can be decreased to every 3 months.270
Although several key molecular events in disease initiation or progression in MM have been confirmed (eg, 14q32 translocations) or implicated
(eg, chromosome 13 deletion), a unifying mechanism of myelomagenesis has eluded investigators. Furthermore, although MM is generally indistinguishable morphologically, it exhibits a tremendous degree of variability clinically with some patients surviving only months and others many years, suggesting that MM is composed of distinct clinical entities.
Gene expression profiling is a powerful tool through which the biology of multiple myeloma can be dissected. Early studies using this technology have provided meaningful insights into myeloma biology, have led to the
identification of new therapeutic targets and have identified powerful
prognostic and pharmacogenomic markers. Expression profiling has also led to the identification of a number of new therapeutic targets not only in myeloma cell survival but also in the pathogenesis of the osteolysis which is a hallmark of this disease.271
In conclusion
Ongoing randomized trials promise to define further the rolesof new agents, mini–allogeneic stem cell transplantation, and maintenancetherapy.
There is a continuing search for active agents basedon advances in
understanding the biology of myeloma. With the recent development and applicationof new genomic technologies (gene expression profiling, array comparative genomic hybridization, single nucleotide polymorphismanalysis), there will be an increasing trend to develop andevaluate therapies based on patient specific genotypic and phenotypicproperties of both the tumor cells and normal cells. In theshort run, these recent advances will help to more preciselyclassify MM that respond most favorably to one or moreof an
ever-widening variety of therapeutic regimens. In thenot too far distant future, however, we are hopeful that ourrapidly increasing understanding of the molecular and cellularbiology of MM, will lead to therapies that directly target differentkinds of MM tumor cells (such as FGFR3 inhibitors for tumorswith t[4;14]) or their requisite interaction with the hostmicroenvironment.
has brought us close to havingthe potential of identifying therapies that will improve rates of complete response, which is aimed at finding a cure either cure MM or convert MM to a chronic disease in a significant fractionof patients with this presently incurable malignancy.
Future research in either direction would be momentous.
References
