Uta Bertsch

Bortezomib Induction and Maintenance Treatment in Patients With Newly Diagnosed Multiple Myeloma: Results of the Randomized Phase III HOVON-65/ GMMG-HD4 Trial

PURPOSE We investigated whether bortezomib during induction and maintenance improves survival in newly diagnosed multiple myeloma (MM). PATIENTS AND METHODS In all, 827 eligible patients with newly diagnosed symptomatic MM were randomly assigned to receive induction therapy with vincristine, doxorubicin, and dexamethasone (VAD) or bortezomib, doxorubicin, and dexamethasone (PAD) followed by high-dose melphalan and autologous stem-cell transplantation. Maintenance consisted of thalidomide 50 mg (VAD) once per day or bortezomib 1.3 mg/m(2) (PAD) once every 2 weeks for 2 years. The primary analysis was progression-free survival (PFS) adjusted for International Staging System (ISS) stage. RESULTS Complete response (CR), including near CR, was superior after PAD induction (15% v 31%; P < .001) and bortezomib maintenance (34% v 49%; P < .001). After a median follow-up of 41 months, PFS was superior in the PAD arm (median of 28 months v 35 months; hazard ratio [HR], 0.75; 95% CI, 0.62 to 0.90; P = .002). In multivariate analysis, overall survival (OS) was better in the PAD arm (HR, 0.77; 95% CI, 0.60 to 1.00; P = .049). In high-risk patients presenting with increased creatinine more than 2 mg/dL, bortezomib significantly improved PFS from a median of 13 months to 30 months (HR, 0.45; 95% CI, 0.26 to 0.78; P = .004) and OS from a median of 21 months to 54 months (HR, 0.33; 95% CI, 0.16 to 0.65; P < .001). A benefit was also observed in patients with deletion 17p13 (median PFS, 12 v 22 months; HR, 0.47; 95% CI, 0.26 to 0.86; P = .01; median OS, 24 months v not reached at 54 months; HR, 0.36; 95% CI, 0.18 to 0.74; P = .003). CONCLUSION Bortezomib during induction and maintenance improves CR and achieves superior PFS and OS.

Administration of bortezomib before and after autologous stem cell transplantation improves outcome in multiple myeloma patients with deletion 17p

In patients with multiple myeloma (MM), risk stratification by chromosomal abnormalities may enable a more rational selection of therapeutic approaches. In the present study, we analyzed the prognostic value of 12 chromosomal abnormalities in a series of 354 MM patients treated within the HOVON-65/GMMG-HD4 trial. Because of the 2-arm design of the study, we were able to analyze the effect of a bortezomib-based treatment before and after autologous stem cell transplantation (arm B) compared with standard treatment without bortezomib (arm A). For allanalyzed chromosomal aberrations, progression-free survival (PFS) and overall survival (OS) were at least equal or superior in the bortezomib arm compared with the standard arm. Strikingly, patients with del(17p13) benefited the most from the bortezomib-containing treatment: the median PFS in arm A was 12.0 months and in arm B it was 26.2 months (P = .024); the 3 year-OS for arm A was 17% and for arm B it was 69% (P = .028). After multivariate analysis, del(17p13) was an independent predictor for PFS (P < .0001) and OS (P < .0001) in arm A, whereas no statistically significant effect on PFS (P = .28) or OS (P = .12) was seen in arm B. In conclusion, the adverse impact of del(17p13) on PFS and OS could be significantly reduced by bortezomib-based treatment, suggesting that long-term administration of bortezomib should be recommended for patients carrying del(17p13).

Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients

To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138(+) plasma cells of 320 newly diagnosed myeloma patients included in the Dutch-Belgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6 corresponded to clusters described in the University of Arkansas for Medical Science (UAMS) classification, CD-1 (n = 13, 4.1%), CD-2 (n = 34, 1.6%), MF (n = 32, 1.0%), MS (n = 33, 1.3%), proliferation-associated genes (n = 15, 4.7%), and hyperdiploid (n = 77, 24.1%). Moreover, the UAMS low percentage of bone disease cluster was identified as a subcluster of the MF cluster (n = 15, 4.7%). One subgroup (n = 39, 12.2%) showed a myeloid signature. Three novel subgroups were defined, including a subgroup of 37 patients (11.6%) characterized by high expression of genes involved in the nuclear factor kappa light-chain-enhancer of activated B cells pathway, which include TNFAIP3 and CD40. Another subgroup of 22 patients (6.9%) was characterized by distinct overexpression of cancer testis antigens without overexpression of proliferation genes. The third novel cluster of 9 patients (2.8%) showed up-regulation of protein tyrosine phosphatases PRL-3 and PTPRZ1 as well as SOCS3. To conclude, in addition to 7 clusters described in the UAMS classification, we identified 3 novel subsets of multiple myeloma that may represent unique diagnostic entities.

Mechanisms of peripheral neuropathy associated with bortezomib and vincristine in patients with newly diagnosed multiple myeloma: a prospective analysis of data from the HOVON-65/GMMG-HD4 trial.

BACKGROUND Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity in patients with multiple myeloma, often requiring adjustment of treatment and affecting quality of life. We investigated the molecular profiles of early-onset (within one treatment cycle) versus late-onset (after two or three treatment cycles) bortezomib-induced peripheral neuropathy and compared them with those of vincristine-induced peripheral neuropathy during the induction phase of a prospective phase 3 trial. METHODS In the induction phase of the HOVON-65/GMMG-HD4 trial, patients (aged 18-65 years) with newly diagnosed Salmon and Durie stage 2 or 3 multiple myeloma were randomly assigned to three cycles of bortezomib-based or vincristine-based induction treatment. We analysed the gene expression profiles and single-nucleotide polymorphisms (SNPs) of pretreatment samples of myeloma plasma cells and peripheral blood, respectively. This study is registered, number ISRCTN64455289. FINDINGS We analysed gene expression profiles of myeloma plasma cells from 329 (39%) of 833 patients at diagnosis, and SNPs in DNA samples from 369 (44%) patients. Early-onset bortezomib-induced peripheral neuropathy was noted in 20 (8%) patients, and 63 (25%) developed the late-onset type. Early-onset and late-onset vincristine-induced peripheral neuropathy was noted in 11 (4%) and 17 (7%) patients, respectively. Significant genes in myeloma plasma cells from patients that were associated with early-onset bortezomib-induced peripheral neuropathy were the enzyme coding genes RHOBTB2 (upregulated by 1·59 times; p=4·5×10(-5)), involved in drug-induced apoptosis, CPT1C (1·44 times; p=2·9×10(-7)), involved in mitochondrial dysfunction, and SOX8 (1·68 times; p=4·28×10(-13)), involved in development of peripheral nervous system. Significant SNPs in the same patients included those located in the apoptosis gene caspase 9 (odds ratio [OR] 3·59, 95% CI 1·59-8·14; p=2·9×10(-3)), ALOX12 (3·50, 1·47-8·32; p=3·8×10(-3)), and IGF1R (0·22, 0·07-0·77; p=8·3×10(-3)). In late-onset bortezomib-induced peripheral neuropathy, the significant genes were SOD2 (upregulated by 1·18 times; p=9·6×10(-3)) and MYO5A (1·93 times; p=3·2×10(-2)), involved in development and function of the nervous system. Significant SNPs were noted in inflammatory genes MBL2 (OR 0·49, 95% CI 0·26-0·94; p=3·0×10(-2)) and PPARD (0·35, 0·15-0·83; p=9·1×10(-3)), and DNA repair genes ERCC4 (2·74, 1·56-4·84; p=1·0×10(-3)) and ERCC3 (1·26, 0·75-2·12; p=3·3×10(-3)). By contrast, early-onset vincristine-induced peripheral neuropathy was characterised by upregulation of genes involved in cell cycle and proliferation, including AURKA (3·31 times; p=1·04×10(-2)) and MKI67 (3·66 times; p=1·82×10(-3)), and the presence of SNPs in genes involved in these processes-eg, GLI1 (rs2228224 [0·13, 0·02-0·97, p=1·18×10(-2)] and rs2242578 [0·14, 0·02-1·12, p=3·00×10(-2)]). Late-onset vincristine-induced peripheral neuropathy was associated with the presence of SNPs in genes involved in absorption, distribution, metabolism, and excretion-eg, rs1413239 in DPYD (3·29, 1·47-7·37, 5·40×10(-3)) and rs3887412 in ABCC1 (3·36, 1·47-7·67, p=5·70×10(-3)). INTERPRETATION Our results strongly suggest an interaction between myeloma-related factors and the patients genetic background in the development of treatment-induced peripheral neuropathy, with different molecular pathways being implicated in bortezomib-induced and vincristine-induced peripheral neuropathy.

Thalidomide in induction treatment increases the very good partial response rate before and after high-dose therapy in previously untreated multiple myeloma

Thalidomide as part of initial treatment of multiple myeloma improves pre- and post-transplant response by increasing the proportion of patients achieving a very good partial response. In the prospective phase 3 HOVON-50/GMMG-HD3 trial, patients randomized to TAD (thalidomide, doxorubicin, dexamethasone) had a significantly higher response rate (at least PR) after induction compared with patients randomized to VAD (vincristine, adriamycin, dexamethasone, 72% vs. 54%, p<0.001). Complete remission (CR) and very good partial remission (VGPR) were also higher after TAD. After High Dose melphalan 200mg/m2 response was comparable in both arms, 76% and 79% respectively. However, CR plus VGPR were significantly higher in the patients randomized to TAD (49% vs. 32%, p<0.001). CTC grade 3–4 adverse events were similar in both arms.

The role of IGF-1 as a major growth factor for myeloma cell lines and the prognostic relevance of the expression of its receptor.

A plethora of myeloma growth factors (MGFs) has been identified, but their relative importance and cooperation have not been determined. We investigated 5 MGFs (interleukin-6 [IL-6], insulin-like growth factor type 1 [IGF-1], hepatocyte growth factor [HGF], HB-epidermal growth factor [HB-EGF], and a proliferation-inducing ligand [APRIL]) in serum-free cultures of human myeloma cell lines (HMCLs). In CD45(-) HMCLs, an autocrine IGF-1 loop promoted autonomous survival whereas CD45(+) HMCLs could not survive without addition of MGFs, mainly IGF-1 and IL-6. IGF-1 was the major one: its activity was abrogated by an IGF-1R inhibitor only, whereas IL-6, HGF, or HB-EGF activity was inhibited by both IGF-1R- and receptor-specific inhibition. APRIL activity was inhibited by its specific inhibitor only. Of the investigated MGFs and their receptors, only expressions of IGF-1R and IL-6R in multiple myeloma cells (MMCs) of patients delineate a group with adverse prognosis. This is mainly explained by a strong association of IGF-1R and IL-6R expression and t(4;14) translocation, but IGF-1R expression without t(4;14) can also have a poor prognosis. Thus, IGF-1-targeted therapy, eventually in combination with anti-IL-6 therapy, could be promising in a subset of patients with MMCs expressing IGF-1R.

Induction of angiogenesis by normal and malignant plasma cells

Abundant bone marrow angiogenesis is present in almost all myeloma patients requiring therapy and correlated to treatment response and survival. We assessed the expression of 402 angiogenesis-associated genes by Affymetrix DNA microarrays in 466 samples, including CD138-purified myeloma cells (MMCs) from 300 previously untreated patients, in vivo microcirculation by dynamic contrast-enhanced magnetic resonance imaging, and in vitro angiogenesis (AngioKit-assay). Normal bone marrow plasma cells (BMPCs) express a median of 39 proangiogenic (eg, VEGFA, ADM, IGF-1) and 28 antiangiogenic genes (eg, TIMP1, TIMP2). Supernatants of BMPCs unlike those of memory B cells induce angiogenesis in vitro. MMCs do not show a significantly higher median number of expressed proangiogenic (45) or antiangiogenic (31) genes, but 97% of MMC samples aberrantly express at least one of the angiogenic factors HGF, IL-15, ANG, APRIL, CTGF, or TGFA. Supernatants of MMCs and human myeloma cell lines induce significantly higher in vitro angiogenesis compared with BMPCs. In conclusion, BMPCs express a surplus of proangiogenic over antiangiogenic genes transmitting to the ability to induce in vitro angiogenesis. Aberrant expression of proangiogenic and down-regulation of antiangiogenic genes by MMCs further increases the angiogenic stimulus, together leading to bone marrow angiogenesis at various degrees in all myeloma patients.

Proliferation is a central independent prognostic factor and target for personalized and risk adapted treatment in multiple myeloma

Background Proliferation of malignant plasma cells is a strong adverse prognostic factor in multiple myeloma and simultaneously targetable by available (e.g. tubulin polymerase inhibitors) and upcoming (e.g. aurora kinase inhibitors) compounds. Design and Methods We assessed proliferation using gene expression-based indices in 757 samples including independent cohorts of 298 and 345 samples of CD138-purified myeloma cells from previously untreated patients undergoing high-dose chemotherapy, together with clinical prognostic factors, chromosomal aberrations, and gene expression-based high-risk scores. Results In the two cohorts, 43.3% and 39.4% of the myeloma cell samples showed a proliferation index above the median plus three standard deviations of normal bone marrow plasma cells. Malignant plasma cells of patients in advanced stages or those harboring disease progression-associated gain of 1q21 or deletion of 13q14.3 showed significantly higher proliferation indices; patients with gain of chromosome 9, 15 or 19 (hyperdiploid samples) had significantly lower proliferation indices. Proliferation correlated with the presence of chromosomal aberrations in metaphase cytogenetics. It was significantly predictive for event-free and overall survival in both cohorts, allowed highly predictive risk stratification (e.g. event-free survival 12.7 versus 26.2 versus 40.6 months, P<0.001) of patients, and was largely independent of clinical prognostic factors, e.g. serum β2-microglobulin, International Staging System stage, associated high-risk chromosomal aberrations, e.g. translocation t(4;14), and gene expression-based high-risk scores. Conclusions Proliferation assessed by gene expression profiling, being independent of serum-β2-microglobulin, International Staging System stage, t(4;14), and gene expression-based risk scores, is a central prognostic factor in multiple myeloma. Surrogating a biological targetable variable, gene expression-based assessment of proliferation allows selection of patients for risk-adapted anti-proliferative treatment on the background of conventional and gene expression-based risk factors.

Combining information regarding chromosomal aberrations t(4;14) and del(17p13) with the International Staging System classification allows stratification of myeloma patients undergoing autologous stem cell transplantation

Background Chromosomal abnormalities have been shown to play a major role in disease evolution of multiple myeloma. Specific changes in interphase cells can be detected by fluorescent in situ hybridization, which overcomes the problem of the lack of dividing cells required for conventional cytogenetics. Design and Methods We analyzed the prognostic value of 12 frequent chromosomal abnormalities detected by fluorescent in situ hybridization in a series of patients (n=315) with newly diagnosed, symptomatic multiple myeloma. All patients underwent frontline autologous stem cell transplantation according to the GMMG-HD3- or GMMG-HD4-trial protocols or analogous protocols. Results Univariate statistical analyses revealed that the presence of del(13q14), del(17p13), t(4;14), +1q21 and non-hyperdiploidy was associated with adverse progression-free and overall survival rates independently of the International Staging System (ISS) classification. Multivariate analyses showed that only t(4;14) and del(17p13) retained prognostic value for both progression-free and overall survival. According to the presence or absence of t(4;14) and del(17p13) and the patients’ International Staging System classification, the cohort could be stratified into three distinct groups: a group with a favorable prognosis [absence of t(4;14)/del(17p13) and ISS I], a group with a poor prognosis [presence of t(4;14)/del(17p13) and ISS II/III] and a group with an intermediate prognosis (all remaining patients). The probabilities of overall survival at 5 years decreased from 72% in the favorable prognostic group to 62% (hazard ratio 2.4; P=0.01) in the intermediate and 41% (hazard ratio 5.6; P<0.001) in the poor prognostic groups. Conclusions These results have implications for risk-adapted management for patients with multiple myeloma undergoing high-dose chemotherapy followed by autologous stem cell transplantation and suggest that new treatment concepts are urgently needed for patients who belong to the poor prognosis group. As targeted therapies evolve, different treatment options might have variable success, depending on the underlying genetic nature of the clone.

Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma

Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.