Thalidomide in total therapy 2 overcomes inferior prognosis of myeloma with low expression of the glucocorticoid receptor gene NR3C1.

PURPOSE: Because dexamethasone remains a key component of myeloma therapy, we wished to examine the impact of baseline and relapse expression levels of the glucocorticoid receptor gene NR3C1 on survival outcomes in the context of treatment with or without thalidomide. EXPERIMENTAL DESIGN: We investigated the clinical impact of gene expression profiling (GEP)-derived expression levels of NR3C1 in 351 patients with GEP data available at baseline and in 130 with data available at relapse, among 668 subjects accrued to total therapy 2 (TT2). RESULTS: Low NR3C1 expression levels had a negative impact on progression-free survival (PFS; HR, 1.47; P = 0.030) and overall survival (OS; HR, 1.90; P = 0.002) in the no-thalidomide arm. Conversely, there was a significant clinical benefit of thalidomide for patients with low receptor levels (OS: HR, 0.54; P = 0.015; PFS: HR, 0.54; P = 0.004), mediated most likely by thalidomide's upregulation of NR3C1. In the context of both baseline and relapse parameters, post-relapse survival (PRS) was adversely affected by low NR3C1 levels at relapse in a multivariate analysis (HR, 2.61; P = 0.012). CONCLUSION: These findings justify the inclusion of NR3C1 expression data in the work-up of patients with myeloma as it can significantly influence the choice of therapy and, ultimately, OS. The identification of an interaction term between thalidomide and NR3C1 underscores the importance of pharmacogenomic studies in the systematic study of new drugs.

Extramedullary disease portends poor prognosis in multiple myeloma and is over-represented in high-risk disease even in the era of novel agents.

BACKGROUND: Extramedullary disease is an uncommon manifestation in multiple myeloma and can either accompany newly diagnosed disease or develop with disease progression or relapse. We evaluated the impact of this disease feature on patients' outcome in the context of novel agents. DESIGN AND METHODS: We analyzed clinical and biological features of extramedullary disease in 936 patients with multiple myeloma enrolled in Total Therapy protocols, 240 patients in non-Total Therapy protocols, and 789 non-protocol patients, all of whom had baseline positron emission tomography scans to document extramedullary disease at diagnosis and its subsequent development at the time of disease progression or relapse. RESULTS: The most common sites for extramedullary disease at diagnosis were skin and soft tissue whereas liver involvement was the striking feature in extramedullary disease at disease relapse or progression. Regardless of therapy, extramedullary disease was associated with shorter progression-free and overall survival, as well as the presence of anemia, thrombocytopenia, elevated serum lactate dehydrogenase, cytogenetic abnormalities, and high-risk features in 70-and 80-gene risk models in univariate analysis. Multivariate analysis with logistic regression revealed that this disease feature was more prevalent in patients with an elevated centrosome index, as determined by gene expression profiling, as well as in myeloma molecular subtypes that are more prone to relapse. These include the MF subtype (also called the "MAF" subtype, associated with over-expression of the MAF gene seen with chromosome translocation 14;16 or 14;20) and the PR subtype (also called the "Proliferation" subtype, associated with overexpression of pro-proliferative genes). CONCLUSIONS: These data show that extramedullary disease is more prevalent in genomically defined high-risk multiple myeloma and is associated with shorter progression-free survival and overall survival, even in the era of novel agents. All clinical trials included in the analyses were registered with www.clinicaltrials.gov (NCT00083551, NCT00083876, NCT00081939, NCT00572169, NCT00644228,NCT00002548,NCT00734877).

Pharmacogenomics of bortezomib test-dosing identifies hyperexpression of proteasome genes, especially PSMD4, as novel high-risk feature in myeloma treated with Total Therapy 3.

Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

International staging system and metaphase cytogenetic abnormalities in the era of gene expression profiling data in multiple myeloma treated with total therapy 2 and 3 protocols.

BACKGROUND: Myeloma survival varies markedly with International Staging System classification, presence of cytogenetic abnormalities, and, especially, gene expression profiling-based risk and delTP53 status, whose collective impact has not been examined in the context of specific therapies. METHODS: The authors examined overall survival (OS), event-free survival (EFS), and complete response duration (CRD) in Total Therapy 2 with randomization to a control or thalidomide arm and in Total Therapy 3 with added bortezomib. Among 612 patients with complete data sets, multivariate analyses were used to investigate the relative contributions to OS, EFS, and CRD of International Staging System stage, cytogenetic abnormalities, and gene expression profiling-derived risk and delTP53 status, in the context of the 3 Total Therapy regimens. RESULTS: Whereas gene expression profiling risk segregated outcomes within all 3 International Staging System stages, International Staging System 3 conferred inferior prognosis only in low-risk myeloma, whereas the grave prognosis of high-risk disease was International Staging System-independent. After adjusting for gene expression profiling-defined high risk and delTP53 in multivariate analysis, International Staging System 3 and cytogenetic abnormalities both retained independent adverse implications for OS, EFS, and CRD. Among the 86% with low-risk disease, cytogenetic abnormalities and delTP53 both affected all 3 endpoints negatively, whereas the International Staging System 3 effect was limited to OS. Total Therapy 3 improved survival outcomes beyond results obtained with Total Therapy 2. CONCLUSIONS: In the genomic era, standard laboratory variables, such as International Staging System stage and presence of cytogenetic abnormalities, continue to impact survival after adjusting for gene expression profiling risk and delTP53 status, providing a basis for stratification in our current practice of gene expression profiling risk-based treatment assignment.

Total Therapy 3 for multiple myeloma: prognostic implications of cumulative dosing and premature discontinuation of VTD maintenance components, bortezomib, thalidomide, and dexamethasone, relevant to all phases of therapy.

The impact of cumulative dosing and premature drug discontinuation (PMDD) of bortezomib (V), thalidomide (T), and dexamethasone (D) on overall survival (OS), event-free survival (EFS), time to next therapy, and post-relapse survival in Total Therapy 3 were examined, using time-dependent methodology, relevant to induction, peritransplantation, consolidation, and maintenance phases. Univariately, OS and EFS were longer in case higher doses were used of all agents during induction, consolidation (except T), and maintenance (except V and T). The favorable OS and EFS impact of D induction dosing provided the rationale for examining the expression of glucocorticoid receptor NR3C1, top-tertile levels of which significantly prolonged OS and EFS and rendered outcomes independent of D and T dosing, whereas T and D, but not V, dosing was critical to outcome improvement in the bottom-tertile NR3C1 setting. PMDD of V was an independent highly adverse feature for OS (hazard ratio = 6.44; P < .001), whereas PMDD of both T and D independently imparted shorter time to next therapy. The absence of adverse effects on postrelapse survival of dosing of any VTD components and indeed a benefit from V supports the use up-front of all active agents in a dose-dense and dose-intense fashion, as practiced in Total Therapy 3, toward maximizing myeloma survival.

High-risk myeloma is associated with global elevation of miRNAs and overexpression of EIF2C2/AGO2.

MicroRNAs (miRNAs) are noncoding RNAs that regulate global gene expression. miRNAs often act synergistically to repress target genes, and their dysregulation can contribute to the initiation and progression of a variety of cancers. The clinical relationship between global expression of miRNA and mRNA in cancer has not been studied in detail. We used whole-genome microarray analyses of CD138-enriched plasma cells from 52 newly diagnosed cases of multiple myeloma to correlate miRNA expression profiles with a validated mRNA-based risk stratification score, proliferation index, and predefined gene sets. In stark contrast to mRNAs, we discovered that all tested miRNAs were significantly up-regulated in high-risk disease as defined by a validated 70-gene risk score (P < 0.01) and proliferation index (P < 0.05). Increased expression of EIF2C2/AGO2, a master regulator of the maturation and function of miRNAs and a component of the 70-gene mRNA risk model, is driven by DNA copy number gains in MM. Silencing of AGO2 dramatically decreased viability in MM cell lines. Genome-wide elevated expression of miRNAs in high-risk MM may be secondary to deregulation of AGO2 and the enzyme complexes that regulate miRNA maturation and function.

Superior results of Total Therapy 3 (2003-33) in gene expression profiling-defined low-risk multiple myeloma confirmed in subsequent trial 2006-66 with VRD maintenance.

The Total Therapy 3 trial 2003-33 enrolled 303 newly diagnosed multiple myeloma patients and was noted to provide superior clinical outcomes compared with predecessor trial Total Therapy 2, especially in gene expression profiling (GEP)-defined low-risk disease. We report here on the results of successor trial 2006-66 with 177 patients, using bortezomib, lenalidomide, and dexamethasone maintenance for 3 years versus bortezomib, thalidomide, and dexamethasone in year 1 and thalidomide/dexamethasone in years 2 and 3 in the 2003-33 protocol. Overall survival (OS) and event-free survival (EFS) plots were super-imposable for the 2 trials, as were onset of complete response and complete response duration (CRD), regardless of GEP risk. GEP-defined high-risk designation, pertinent to 17% of patients, imparted inferior OS, EFS, and CRD in both protocols and, on multivariate analysis, was the sole adverse feature affecting OS, EFS, and CRD. Mathematical modeling of CRD in low-risk myeloma predicted a 55% cure fraction (P < .001). Despite more rapid onset and higher rate of CR than in other molecular subgroups, CRD was inferior in CCND1 without CD20 myeloma, resembling outcomes in MAF/MAFB and proliferation entities. The robustness of the GEP risk model should be exploited in clinical trials aimed at improving the notoriously poor outcome in high-risk disease.

TP53 deletion is not an adverse feature in multiple myeloma treated with total therapy 3.

Contrary to Total Therapy (TT) 2 for multiple myeloma patients, FGFR3- translocation bore no adverse effects on outcome in TT3 with added bortezomib. Del TP53, another poor-risk feature in TT2 and present in 10% of 441 patients treated, was examined for its prognostic consequences in TT3. Not affecting rate or duration of complete response, TP53 haplo-insufficiency also did not compromise, in the 83% with genomically defined low-risk myeloma, survival or event-free survival. FGFR3+ and FGFR3- molecular subgroups fared worse in the presence of del TP53 when applying TT2 but not TT3. Thus, the prognostic implications of del TP53 were protocol-, genome-defined risk- and molecular subgroup-dependent.

Complete remission in multiple myeloma examined as time-dependent variable in terms of both onset and duration in Total Therapy protocols.

Landmark analyses are used to investigate the importance for survival of achieving complete response (CR), an important initial goal of myeloma therapy. With median times to CR in Total Therapy (TT) trials of approximately 1 year, this approach excludes a sizeable fraction of patients dying before such a landmark. To permit inclusion of all trial participants, we investigated the prognostic implications of both onset and duration of CR as time-dependent variables. Superseding the adverse effects of cytogenetic abnormalities and other standard prognostic parameters, both failure to achieve CR (non-CR) and, especially, loss of CR (los-CR) were independently associated with inferior survival in TT1, TT2, and TT3 protocols. In the context of gene array-defined risk, available in TT2 and TT3 subsets, both los-CR and non-CR terms were retained in the survival model as dominant adverse variables, stressing the prognostic importance of sustaining CR status, especially in high-risk disease.

Gene expression profiling of plasma cells at myeloma relapse from tandem transplantation trial Total Therapy 2 predicts subsequent survival.

We report on prognostic implications for post-relapse survival (PRS) of a gene expression profiling (GEP)-defined risk score at relapse available in 120 myeloma patients previously enrolled in tandem transplantation trial Total Therapy 2. Among the 71 patients with additional GEP baseline information, 3-year PRS was 71% in 40 patients with low risk present both at baseline and relapse contrasting with only 17% in 28 patients with high risk at relapse, 12 of whom with baseline low-risk status fared better than the remainder (P = .08). On multivariate analysis of relapse parameters available in 104 patients, high risk conferred short PRS (hazard ratio = 4.00, P < .001, R(2) = 33%), whereas relapse hyperdiploidy predicted long PRS (hazard ratio = 0.37, P = .022, cumulative R(2) = 41%). In case the initial partial response lasted less than 2 years, relapse low-risk identified 26 patients with superior 3-year PRS of 61% versus 9% among 32 with relapse high-risk (P < .001). Based on its PRS predictive power, GEP analysis should be an integral part of new agent trials in search of better therapy for high-risk myeloma.

Seven-year median time to progression with thalidomide for smoldering myeloma: partial response identifies subset requiring earlier salvage therapy for symptomatic disease.

Smoldering multiple myeloma (SMM) is usually followed expectantly without therapy. We conducted a phase 2 trial in 76 eligible patients with SMM, combining thalidomide (THAL, 200 mg/d) with monthly pamidronate. In the first 2 years, THAL dose reduction was required in 86% and drug was discontinued in 50%. Within 4 years, 63% improved, including 25% qualifying for partial response (PR); by then, 34 patients had progressed and 17 required salvage therapy. Unexpectedly, attaining PR status was associated with a shorter time to salvage therapy for disease progression (P < .001), perhaps reflecting greater drug sensitivity of more aggressive disease. Low beta-2-microglobulin levels less than 2 mg/L were independently associated with superior overall and event-free survival. Four-year survival and event-free survival estimates of 91% and 60%, respectively, together with a median postsalvage therapy survival of more than 5 years justify the conduct of a prospective randomized clinical trial to determine the clinical value of preemptive therapy in SMM. Trial registered at http://www.clinicaltrials.gov under identifier NCT00083382.

Proteomics analysis in post-transplant lymphoproliferative disorders.

Little is known about the biology of post-transplant lymphoproliferative disorders (PTLDs). The objective of this study was to determine the molecular alterations that occur at the protein level in patients with PTLDs. Six tumor samples from adult patients with PTLD and four benign lymph nodes were studied using protein microarray technique. Proteins that were dysregulated included proteins in the PI3K/mTOR, NFkB and HSP90 pathways. Inhibitors of these proteins induced cytotoxicity and apoptosis in EBV+ve and -ve cell lines. These results provide insight into pathways that are dysregulated in PTLD and can be targeted in future clinical trials.

Thalidomide arm of Total Therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities.

Total Therapy 2 examined the clinical benefit of adding thalidomide up-front to a tandem transplant regimen for newly diagnosed patients with multiple myeloma. When initially reported with a median follow-up of 42 months, complete response rate and event-free survival were superior among the 323 patients randomized to thalidomide, whereas overall survival was indistinguishable from that of the 345 patients treated on the control arm. With further follow-up currently at a median of 72 months, survival plots segregated 5 years after initiation of therapy in favor of thalidomide (P = .09), reaching statistical significance for the one third of patients exhibiting cytogenetic abnormalities (CAs; P = .02), a well-recognized adverse prognostic feature. The duration of complete remission was also superior in the cohort presenting with CAs such that, at 7 years from onset of complete remission, 45% remained relapse-free as opposed to 20% on the control arm (P = .05). These observations were confirmed when examined by multivariate analysis demonstrating that thalidomide reduced the hazard of death by 41% among patients with CA-positive disease (P = .008). Because two thirds of patients without CAs have remained alive at 7 years, the presently emerging separation in favor of thalidomide may eventually reach statistical significance as well.

Complete remission sustained 3 years from treatment initiation is a powerful surrogate for extended survival in multiple myeloma.

BACKGROUND: Complete response (CR) has been considered a necessary although not sufficient early clinical endpoint for extended survival in multiple myeloma. METHODS: By using Total Therapy 2 (TT2) clinical outcome data in 668 patients, whether sustained CR (SUS-CR) was potentially a superior surrogate for survival than attaining CR status per se was evaluated. RESULTS: Compared with not achieving CR (NON-CR) and especially achieving and subsequently losing CR status (LOS-CR) within a 3-year landmark from treatment initiation, SUS-CR was associated with highly superior postlandmark survival (P < .0001). These results were validated in 231 untreated patients enrolled in the predecessor trial, TT1 (hazard ratio [HR] = 0.54, P = .013) and in 1103 previously treated patients on other transplant protocols (HR = 0.49; P < .001). CONCLUSIONS: In all 3 trial settings the survival benefit of SUS-CR was independent of metaphase abnormalities as a dominant adverse parameter. Given its bleak prognosis despite high CR rates, SUS-CR should be evaluated as a primary trial endpoint in high-risk myeloma.

Targeting Akt and heat shock protein 90 produces synergistic multiple myeloma cell cytotoxicity in the bone marrow microenvironment.

PURPOSE: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. EXPERIMENTAL DESIGN: MM cell lines were incubated with perifosine (5 and 10 micromol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. RESULTS: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. CONCLUSIONS: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Cytogenetically defined myelodysplasia after melphalan-based autotransplantation for multiple myeloma linked to poor hematopoietic stem-cell mobilization: the Arkansas experience in more than 3,000 patients treated since 1989.

Myelodysplastic syndrome (MDS) is a well-recognized complication of chemotherapy for multiple myeloma (MM). Serial bone marrow metaphase examinations were performed for MM restaging in 3,077 patients undergoing high-dose therapy (HDT). MDS-associated cytogenetic abnormalities (MDS-CAs) were observed in 105 of 2,418 patients in whom cytogenetic data were available after HDT. MDS-CAs occurred transiently in 72 patients and on 3 successive occasions (persistent MDS-CAs) in 33 patients, for 10-year estimates of 4% and 2%, respectively; only 21 patients developed overt clinical MDS and 5, acute myeloblastic leukemia (AML). MDS-CA development was linked to lower CD34 yield at collection, longer time interval from MM diagnosis to HDT, older age, and lower platelet recovery after HDT; persistent MDS-CAs were predicted by CD34 yield of less than 3 x 10(6)/kg and need for more than 2 apheresis procedures. Applying a tertile frequency distribution over time to all 105 patients with MDS-CAs, its detection early after HDT was associated with longer time interval from diagnosis and low pre-HDT platelet count (likely resulting from pre-HDT damage), whereas late-onset MDS-CAs were noted among patients treated with Total Therapy 2 and Total Therapy 3 that applied post-HDT consolidation chemotherapy (suggesting possible post-HDT damage). While the risk of MDS-CAs was low and clinical MDS occurred infrequently, monitoring after post-HDT consolidation chemotherapy appears warranted.

Benefit of complete response in multiple myeloma limited to high-risk subgroup identified by gene expression profiling.

EXPERIMENTAL DESIGN: To determine whether the clinical benefit of complete remission (CR) may depend on prognostic subgroups of patients with multiple myeloma. PATIENTS AND METHODS: Newly diagnosed patients with myeloma received a tandem autotransplant regimen. Using multivariate regression analyses, we examined the prognostic implications of time-dependent onset of CR on overall survival and event-free survival in the context of standard prognostic factors (SPF) and gene expression profiling-derived data available for 326 patients. RESULTS: CR benefited patients regardless of risk status when only SPFs were examined. With knowledge of gene array data, a survival (and event-free survival) benefit of CR only pertained to the small high-risk subgroup of 13% of patients (hazard ratio, 0.23; P = 0.001), whereas the majority of patients with low-risk disease had similar survival expectations whether or not CR was achieved (hazard ratio, 0.68; P = 0.128). CONCLUSIONS: Access to gene expression information permitted the recognition of a small very high-risk subgroup of 13% of patients, in whom prolonged survival critically depended on achieving CR. Absence of such benefit in the remainder should lead to a reassessment of clinical trial designs that rely on this end point as a surrogate for long-term prognosis.

Incorporating bortezomib into upfront treatment for multiple myeloma: early results of total therapy 3.

Total therapy 3 incorporated bortezomib into a melphalan-based tandem transplant regimen for 303 newly diagnosed patients with myeloma. Induction chemotherapy prior to and consolidation chemotherapy after transplants each consisted of two cycles of VTD-PACE (bortezomib, thalidomide, dexamethasone and 4-d continuous infusions of cis-platin, doxorubicin, cyclophosphamide, etoposide); 3-year maintenance comprised monthly cycles of VTD in the first and TD in the remaining years. The median age was 59 years (age >64 years, 28%). A minimum of 20 x 10(6) CD34 cells/kg was collected in 87% of patients; 83% completed both transplants, and only 5% suffered a treatment-related death. At 24 months, 83% had achieved near-complete remission, which was sustained in 88% at 2 years from its onset. With a median follow-up of 20 months, 2-year estimates of event-free and overall survival were 84% and 86% respectively. The 44 patients who experienced an event more often had a high-risk gene array profile, cytogenetic abnormalities and indicators of high lactate dehydrogenase, beta-2-microglobulin, creatinine and International Staging System stage. Toxicities of grade > 2 included thrombo-embolic events in 27% and peripheral neuropathy in 12%. Results of this phase-2 study demonstrated that bortezomib could be safely combined with multi-agent chemotherapy, effecting near-complete remission status and 2-year survival rates in more than 80% of patients.

Testing standard and genetic parameters in 220 patients with multiple myeloma with complete data sets: superiority of molecular genetics.

Prognostic models for multiple myeloma have been fraught with tremendous heterogeneity in outcome among subgroups. In the context of Total Therapy 2, a tandem transplant trial for newly diagnosed myeloma, comprehensive information was available in 220 patients on standard prognostic factors (SPF), magnetic resonance imaging (MRI)-defined focal lesions, cytogenetic abnormalities (CA), fluorescence-in-situ-hybridisation (FISH)-derived amplification of chromosome 1q21 (amp1q21) and deletion of 13q14, as well as gene expression profiling (GEP). Five multivariate analysis-based survival models were derived, utilising SPF only (model 1), with progressive addition of CA (model 2), MRI (model 3), FISH (model 4) and GEP (model 5). The R(2) value, a measure of accounting for clinical outcome variability, increased progressively from 18% in model 1 to 38% in model 5. The hazard ratio for overall survival was highest for GEP (3.07, P < 0.001) followed by amp1q21 (1.71, P = 0.05). According to the presence of none (49%), one (35%) or both of these two risk features (16%), 3-year survival decreased progressively from 92% to 78% to 43% (P < 0.0001). Thus, the dominance over other prognostic parameters of molecular genetics justifies the generation of quantitative reverse transcription polymerase chain reaction methodology ('MM genetic kit') for the optimal risk stratification of patients participating in therapeutic trials.