Bortezomib with Thalidomide plus Dexamethasone Compared with Thalidomide plus Doxorubicin and Dexamethasone as Induction Therapy in Previously Untreated Multiple Myeloma Patients

High-dose therapy plus autologous stem cell transplantation (HDT/ASCT) is commonly considered the standard of care for frontline treatment in younger patients with multiple myeloma (MM) [1]. In the context of HDT/ASCT, achievement of complete response (CR) or at least very good partial response (VGPR) is associated with an improved outcome [2,3,4,5]. One strategy to increase the CR plus VGPR rate in the HDT/ASCT paradigm is to improve induction treatment. Before the era of novel therapies, induction treatment typically consisted of high-dose dexamethasone alone or combined with vincristine and adriamycin (VAD). Compared with dexamethasone or VAD, the combination of thalidomide and dexamethasone as induction treatment increased the overall response rate but failed to increase the CR rate before HDT/ASCT or the CR plus VGPR rate after ASCT [6,7]. A randomized trial showed that compared with VAD, bortezomib plus dexamethasone (VD) significantly improved CR plus VGPR before HDT/ASCT [8]. A three-drug combination including one novel agent, such as thalidomide plus doxorubicin and dexamethasone (TAD), has also been found to be superior to VAD or VAD-like regimens. Recently, the most promising results have been obtained with a three-drug regimen consisting of thalidomide, bortezomib and dexamethasone (VTD) [9,10,11]. The objective of any new induction regimen should be to provide best efficacy while limiting toxicity. The high rate of CR effected by novel agents as induction therapy has also renewed interest in the evaluation of minimal residual disease (MRD) after these combined treatment strategies [12]. In our hospital, we conducted a retrospective study to compare VTD with TAD. Until now, first-line treatment with these combinations has not been reported in any comparative study. The principal objective of this study was to determine whether the combination of VTD would improve the CR and CR plus VGPR rates compared with TAD. Secondary, using additional methods, such as flow cytometric (FC) assays and polymerase chain reaction (PCR) technology, we evaluated the MRD in the subgroup of patients that obtained CR.

We retrospectively evaluated 87 MM patients treated in our department between February 2007 and September 2011 eligible for autologous transplant. Patients were 29–73 years of age (median 61) and had untreated, newly diagnosed and symptomatic MM. Evaluation of disease was based on a variety of laboratory techniques, including bone marrow biopsy, morphology, immunophenotyping, molecular IgH rearrangements, analysis of serum or urine M-component and free light chains (paraprotein in serum >1 g/dl or urine >0.2 g/24 h), hematological and biochemical parameters and cytogenetics. All patients provided written informed consent in accordance with the International Conference on Harmonization of Good Clinical Practice Guidelines and the principles of the Declaration of Helsinki. Patients were treated with 4 cycles of TAD or VTD (TAD if patients were treated between February 2007 and August 2009, and VTD if treated between September 2009 and 2011). TAD consisted of 4 cycles of intravenous doxorubicin on day 1 every 28 days in day hospital, dexamethasone 40 mg orally on days 1–4 and 9–12, and thalidomide 100 mg/day continuously and orally administered. VTD consisted of four 3-week cycles of bortezomib 1.3 mg/m² administered intravenously on days 1, 4, 8 and 11 plus dexamethasone 40 mg on days 1 and 2 as well as on days 3 and 4, 8 and 9, and 11 and 12 (all cycles), and thalidomide 100 mg/day continuously and orally administered. Recommended concomitant medications included bisphosphonates, antibiotics, erythroid or granulocytic growth factor and antiviral prophylaxis in accordance with standard practice. After induction, in respondent patients, stem cells were mobilized with cyclophosphamide 3 g/m² plus granulocyte colony-stimulating factor 10 µg/kg. The target yield was 3.5 × 10⁶ CD34+ cells/kg. The primary endpoint was postinduction CR plus VGPR rates. The secondary endpoint was evaluation of MRD in patients that obtained CR. Response was evaluated according to the International Myeloma Working Group uniform criteria [13]. Adverse events were graded by the National Cancer Institute Common Toxicity Criteria, version 3.0. Blood and 24-hour urine samples were taken at baseline, before each induction cycle and 1 month after cycle 4. FC assays and PCR, on bone marrow samples, were performed at diagnosis and 1 month after cycle 4. The endpoint targets (postinduction CR and VGPR rates) were performed using a χ² test. For the postinduction CR and CR plus VGPR rates, prognostic factors for response were investigated using logistic regression analysis, adjusted for treatment to check whether the absence or the presence of a difference between the response rates for VTD and TAD was confirmed when taking into account factors significantly related to response. Specifically, the following factors were tested: β₂-microglobulin level and cytogenetics.

Immunophenotyping was carried out by a FacsCanto II cytometer equipped with 3 lasers (405, 488 and 633 nm). A seven-color method was used, with monoclonal antibodies conjugated with the following fluorochromes: FITC, PE, PercCP-Cy5.5, Pe-Cy.7, APC, APC-Cy.7 and AmCyan.

Bone marrow samples were stained with 1 µg of each monoclonal antibody and a lyse/no-wash method was carried out after incubation. A total of 500,000 events/tube were acquired and plasma cells were identified by means of a CD138 versus CD38 analysis, which provided the best separation of plasma cells from other leukocytes. A second gate included events with homogeneous forward scatter and side scatter properties, and a third gate included CD38+ events after analysis of a CD38/CD45 dot plot. The three gates were intersected to obtain a well-defined plasma cell population [14,15]. Abnormal plasma cells were defined as CD19– and clonality assessment by cytoplasmic immunoglobulin ĸ or λ light chains. Negativity for CD27 and CD81 and/or positivity for CD20, CD10, CD117 and CD56 were useful additional markers of abnormality [16]. A cluster of at least 50 events was considered significant, and MRD was set at 0.01% abnormal plasma cells [17]. Data were analyzed by the FacsDiva software.

PCR analyses were performed on mononuclear cells separated by Ficoll/Hypaque gradient. High-molecular-weight DNA was extracted, and suitable aliquots were utilized for PCR assays to identify bone marrow infiltration represented by clonal IgH rearrangement. Every PCR procedure included both negative (distilled water) and positive control (DNA carrying monoclonal IgH rearrangement). To reduce false-negative results, six independent IgH PCRs were performed using V_H family-specific sense primers and a JH consensus primer, as previously described by Willems et al. [18]. Samples were prepared for analysis by mixing 1 µl of PCR products with 15 µl of deionized formamide (Amresco) and 0.5 µl Gene Scan™ 500 Tamra-labeled internal standard (Applera). The mixture was denatured at 95°C for 2 min and then quickly cooled on ice. Capillary electrophoresis and fluorescence detection with a virtual filter C was performed using an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Runs were executed with the module GS STR POP 4 (1 ml) C, with 10-second and 15-kV injection and run voltage, 60°C constant temperature, 24-min run time, using polymer POP 4 and the running buffer Genetic Analyzer 1X (Applied Biosystems). Genescan 2.1 software was then used to analyze the PCR products, with accurate sizing and quantification of the peak areas, according to our previously published method [19].

A total of 87 patients were treated and received TAD (44 patients) or VTD (43 patients). Baseline characteristics are summarized in table 1. No significant difference was observed between the two groups. Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in >90% of patients. Both VTD and TAD regimens were well tolerated. Rates of CR and VGPR or better were significantly higher after induction therapy with VTD than with TAD (table 2). Only 1 patient treated with VTD had disease progression during induction therapy. After 4 cycles, the overall response rate was 91% in the VTD group versus 84% in the TAD group (table 2). The difference in the overall response rate between the VTD and TAD arms was 7%. However, the CR plus VGPR rate was significantly higher in the VTD arm (51 vs. 18%; p = 0.001). The difference in the CR plus VGPR rates between the VTD and TAD arms was 33%. The difference in the CR rate was 28% (30% in the VTD group vs. 2% in the TAD group). In the only patient that obtained CR in the TAD group, FC and PCR were able to still detect MRD, but in 10 of 13 (77%) patients that achieved CR in the VTD group, both additional assays confirmed the absence of MRD. The use of cyclophosphamide to mobilize stem cells did not upgrade the response to induction therapy in either arm of the study. None of the analyzed factors mentioned above was predictive for CR plus VGPR.

In MM, the goal of induction treatment before HDT/ASCT is to achieve the highest possible response rate while avoiding impairment of stem cell collection and significant toxicity that could preclude intensive therapy [20]. A small fraction of patients unexpectedly lose their CR status during the first year after HDT/ASCT and experience a dismal survival rate. VD is frequently considered the cornerstone of induction, and several triplet combinations have been developed based on this backbone, such as VD plus doxorubicin, VD plus cyclophosphamide, VD plus thalidomide (VTD), or VD plus lenalidomide [21]. Recently, VTD has been prospectively compared with TD [12] and with VBMCP/VBAP/bortezomib [13], and the efficacy results of these 2 studies are in favor of VTD. Until recently, no direct comparison of TAD versus VTD was available. In our study, patients with previously untreated myeloma who were eligible for transplant, induction therapy with VTD significantly improved clinical outcomes compared with TAD therapy in patients eligible for autologous stem cell transplantation.

The CR plus VGPR rate after 4 cycles was significantly increased in the VTD arm compared with the TAD arm. Several studies have already demonstrated that achievement of VGPR before ASCT is an important prognostic factor and is therefore a key objective, and that the choice of best induction therapy is of great importance [2,3,5,22]. Our VTD efficacy results are in line with those achieved in prospective studies conducted by Italian and Spanish groups. In the Italian prospective study, the VTD regimen was compared to TD. After induction therapy, CR or near CR was achieved in 73 patients (31%) receiving VTD, and in 27 (11%) of the TD group (p < 0.0001) [23]. In the 3-arm randomized Spanish study (PETHEMA GEM05MENOS65), 6 cycles of VTD as induction were compared with 6 cycles of TD, and with 4 cycles of VBMCP/VBAP followed by 2 cycles of bortezomib. VTD yielded a 60% VGPR rate versus 29% in the TD arm and 36% in the third arm of the trial [12]. In our study, both VTD and TAD regimens were well tolerated, with doses during induction and consolidation therapy very close to the planned doses. Side effects, including peripheral neuropathy were manageable during induction therapy. These data also suggest that the combination of bortezomib with thalidomide does not result in additive neurological toxic effects. The high rate of CR effected by novel agents as induction therapy in MM has renewed interest in the evaluation of MRD after these combined treatment strategies, and the VTD regimen seems to be able to induce a very high rate of CR including undetected MRD evaluated with two different analyses. Incorporation of novel agents in induction therapy of patients with myeloma has improved the CR and VGPR rates. In conclusion, our study shows that VTD is a superior induction regimen compared with TAD, with a higher response rate after induction, translating into a greater CR plus VGPR. Additionally, we have shown that when highly sensitive techniques are used, MRD is undetectable in an important fraction of patients in CR after VTD. Therefore, VTD represents a new standard of care to maximize the degree and speed of tumor reduction in patients with myeloma who are eligible for transplant.

G.B. and E.O. contributed equally to this work.