Marina Martello

PET/CT Improves the Definition of Complete Response and Allows to Detect Otherwise Unidentifiable Skeletal Progression in Multiple Myeloma

Purpose: To evaluate the role of 18F-FDG PET/CT in 282 symptomatic multiple myeloma patients treated up-front between 2002 and 2012. Experimental Design: All patients were studied by PET/CT at baseline, during posttreatment follow-up, and at the time of relapse. Their median duration of follow-up was 67 months. Results: Forty-two percent of the patients at diagnosis had >3 focal lesions, and in 50% SUVmax was >4.2; extramedullary disease was present in 5%. On multivariate analysis, ISS stage 3, SUVmax >4.2, and failure to achieve best complete response (CR) were the leading factors independently associated with shorter progression-free survival (PFS) and overall survival (OS). These 3 variables were used to construct a prognostic scoring system based on the number of risk factors. After treatment, PET/CT negativity (PET-neg) was observed in 70% of patients, whereas conventionally defined CR was achieved in 53%. Attainment of PET-neg favorably influenced PFS and OS. PET-neg was an independent predictor of prolonged PFS and OS for patients with conventionally defined CR. Sixty-three percent of patients experienced relapse or progression; in 12%, skeletal progression was exclusively detected by systematic PET/CT performed during follow-up. A multivariate analysis revealed that persistence of SUVmax >4.2 following first-line treatment was independently associated with exclusive PET/CT progression. Conclusions: PET/CT combined with ISS stage and achievement or not of CR on first-line therapy sorted patients into different prognostic groups. PET/CT led to a more careful evaluation of CR. Finally, in patients with persistent high glucose metabolism after first-line treatment, PET/CT can be recommended during follow-up, to screen for otherwise unidentifiable progression. Clin Cancer Res; 21(19); 4384–90. ©2015 AACR.

Bortezomib- and thalidomide-induced peripheral neuropathy in multiple myeloma: clinical and molecular analyses of a phase 3 study

A subanalysis of the GIMEMA‐MMY‐3006 trial was performed to characterize treatment‐emergent peripheral neuropathy (PN) in patients randomized to thalidomide‐dexamethasone (TD) or bortezomib‐TD (VTD) before and after double autologous transplantation (ASCT) for multiple myeloma (MM). A total of 236 patients randomized to VTD and 238 to TD were stratified according to the emergence of grade ≥2 PN. Gene expression profiles (GEP) of CD138+ plasma cells were analyzed in 120 VTD‐treated patients. The incidence of grade ≥2 PN was 35% in the VTD arm and 10% in the TD arm (P < 0.001). PN resolved in 88 and 95% of patients in VTD and TD groups, respectively. Rates of complete/near complete response, progression‐free and overall survival were not adversely affected by emergence of grade ≥2 PN. Baseline characteristics were not risk factors for PN, while GEP analysis revealed the deregulated expression of genes implicated in cytoskeleton rearrangement, neurogenesis, and axonal guidance. In conclusion, in comparison with TD, incorporation of VTD into ASCT was associated with a higher incidence of PN which, however, was reversible in most of the patients and did not adversely affect their outcomes nor their ability to subsequently receive ASCT. GEP analysis suggests an interaction between myeloma genetic profiles and development of VTD‐induced PN. Am. J. Hematol. 89:1085–1091, 2014.

Therapeutic targeting of hypoxia and hypoxia-inducible factor 1 alpha in multiple myeloma.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by accumulation of malignant plasma cells (PCs) within the bone marrow (BM). The PCs are in close contact with stromal cells, which secrete growth factors and cytokines, promoting tumor cell growth and survival. Despite the availability of new drugs with immunomodulatory properties targeting the neoplastic clone and its microenvironment, MM is still an incurable disease, with patients experiencing subsequent phases of remission and relapse, eventually leading to disease resistance and patient death. It is now well established that the MM BM microenvironment is hypoxic, a condition required for the activation of the hypoxia-inducible factor 1 alpha (HIF-1α). It has been shown that HIF-1α is constitutively expressed in MM even in normoxic conditions, suggesting that HIF-1α suppression might be part of a therapeutic strategy. Constitutively activated HIF-1α enhances neovascularization, increases glucose metabolism, and induces the expression of antiapoptotic proteins. HIF-1α is thought to be one of the most important molecular targets in the treatment of cancer, and a variety of chemical inhibitors for HIF-1α have been developed to date. This review examines the role of HIF-1α in MM and recent developments in harnessing the therapeutic potential of HIF-1α inhibition in MM.

HIF-1α inhibition blocks the cross talk between multiple myeloma plasma cells and tumor microenvironment

Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.

Prognostic impact of serial measurements of serum-free light chain assay throughout the course of newly diagnosed multiple myeloma treated with bortezomib-based regimens

Abstract We retrospectively investigated the role of serial serum-free light chain (sFLC) evaluations in 150 multiple myeloma (MM) patients treated with first-line bortezomib-based regimens. Baseline sFLC ratio (sFLCR) identified three groups of patients – normal, lightly abnormal (<100), and highly abnormal (≥100) – with different progression-free survival (PFS: 3-year estimate 72% versus 61% versus 44%, respectively, p = 0.03). Moreover, the achievement of a normal sFLCR correlated with extended PFS (49 versus 17 months, p < 0.0001) and overall survival (75 versus 43 months, p < 0.0001) as compared with abnormal sFLCR, a gain maintained in a multivariate analysis for PFS. At relapse, a high sFLCR was associated with earlier start of salvage therapy compared with sFLCR <100 (3-month probability: 89% versus 64%, p = 0.0426). In 20% of patients, sFLC escape preceded the conventional relapse by a median of 3.8 months. Our results highlight the role of sFLC assay in the prognosis and follow-up of MM.

Opposite activation of the Hedgehog pathway in CD138+ plasma cells and CD138−CD19+ B cells identifies two subgroups of patients with multiple myeloma and different prognosis

Hyperactivation of the Hedgehog (Hh) pathway, which controls refueling of multiple myeloma (MM) clones, might be critical to disease recurrence. Although several studies suggest the Hh pathway is activated in CD138− immature cells, differentiated CD138+ plasma cells might also be able to self-renew by producing themselves the Hh ligands. We studied the gene expression profiles of 126 newly diagnosed MM patients analyzed in both the CD138+ plasma cell fraction and CD138−CD19+ B-cell compartment. Results demonstrated that an Hh-gene signature was able to cluster patients in two subgroups characterized by the opposite Hh pathway expression in mature plasma cells and their precursors. Strikingly, patients characterized by Hh hyperactivation in plasma cells, but not in their B cells, displayed high genomic instability and an unfavorable outcome in terms of shorter progression-free survival (hazard ratio: 1.92; 95% confidence interval: 1.19–3.07) and overall survival (hazard ratio: 2.61; 95% confidence interval: 1.26–5.38). These results suggest that the mechanisms triggered by the Hh pathway ultimately led to identify a more indolent vs a more aggressive biological and clinical subtype of MM. Therefore, patient stratification according to their molecular background might help the fine-tuning of future clinical and therapeutic studies.

Current and emerging triplet combination therapies for relapsed and refractory multiple myeloma

ABSTRACT Despite significant improvement in outcomes have been observed for multiple myeloma (MM) patients over the past 10–15 years, mainly due to the introduction of novel agents targeting the tumor clone and the bone marrow microenvironment, treatment of refractory and/or relapsed (RR) disease remains a challenge, particularly for patients who have failed prior bortezomib- and lenalidomide-based therapies. More recently, new drugs with different mechanisms of action, including second generation proteasome inhibitors, third generation immunomodulatory drugs, histone deacetylase inhibitors and monoclonal antibodies, have been developed and are under investigation, further increasing treatment options for RRMM patients. Overall, novel agent-based triplet combinations demonstrated superior response rates and prolonged disease control when compared with two-drug regimens in several randomized clinical trials, without adding any relevant additional toxicity. Salvage triplet therapies are likely to play a key role in overcoming drug-resistance and hold promise to further improve long-term outcomes of RRMM patients.

Treatment optimization for Multiple Myeloma: schedule-dependent synergistic cytotoxicity of pomalidomide and carfilzomib in an in vitro and ex-vivo model

Proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), significantly enhanced the depth of response and prolonged the survival of Multiple Myeloma (MM) patients, although not all patients respond favourably to treatment. Optimization of treatment schedules and dosages of IMiDs and PIs might lead to improved treatment efficacy. In this study we aimed at exploring the optimal schedule of IMiDs and PIs in both in vitro models, including bone marrow (BM) microenvironment simulation, and an ex-vivo model using patient-derived BM samples. MM cells were exposed to IMiDs and PIs either simultaneously or sequentially. Using the median effect method of Chou Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. We demonstrated schedule-dependent synergistic cytotoxicity for the combination of IMiDs and PIs and a maximal apoptosis was observed in IMiDs pre-exposure schedule. The superior activity of this schedule was maintained even in BM microenvironment models, and was further confirmed using patient-derived samples. Our data overall suggest that the administration of IMiDs before PIs can improve treatment efficacy. Clinical trials are needed to investigate the most effective schedule, which could be to start the administration of IMiDs before PIs to increase cells killing.

Abstract 3189: The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome

The iperactivation of Hedgehog (Hh) pathway, which controls the refuel of the tumor clone, might be critical to Multiple Myeloma (MM) recurrence. In order to dissect the role played by Hh pathway in different MM cells compartments, and to evaluate its clinical impact on patient outcomes, here we explore the transcriptomic and genomic profiles in both CD138+ plasma cells and CD138-19+ B progenitors cells obtained from newly diagnosed MM patients (pts), The study included a cohort of 126 pts, homogenously treated with bortezomib-based regimens and ASCT. DNA and RNA were obtained from both CD138+ plasma cell fraction and CD19+ B cells. Gene expression profiling (GEP) (HG U133 Plus 2.0) and genomic analysis (SNP 6.0) were performed on Affymetrix platform. Data were analysed by employing several software: dChip (GEP clustering), Ingenuity Pathway Analysis (GEP Enrichment) and Nexus (Copy number). By unsupervised hierarchical clustering, an Hh signature of 10 genes - SHH, IHH, DHH, SMO, PTCH1, PTCH2, SUFU, GLI1, GLI2 and GLI3 - was identified, and it was able to significantly cluster pts in two subgroups: cluster 1 (70 pts) and cluster 2 (56 pts. An overall significant activation of Hh pathway was shown in cluster 2, as compared to cluster 1. Of note, the Hh pathway was down regulated in CD19+ B cells obtained from pts included in cluster 2, while it was overexpressed in cluster 1 pts. Western blots on both cell fractions confirmed this opposite Hh genes behavior. A higher genomic instability (e.g. higher frequencies of both t(4;14) and del(17p)) was demonstrated in CD138+ cells from cluster 2 pts and, at least 5 known tumor suppressor genes, such as RB1, BRCA2, PDX1, FOXO1 and TP53 were affected. Conversely, cluster 1 pts were mainly characterized by hyperdiploid karyotypes. The more aggressive phenotype of cluster 2 pts was confirmed by an overall deregulation of cell adhesion processes (CD44, LIMS1, COL4A2, CTGF, COL1A1, FN1), increased proliferation (MYCBP, IL22, SDPR, SOX2, SOX6) and DNA repair mechanisms (SP1, SMARCD3, FOXA3). Hh pathway activation significantly influenced pts’ outcome, since those included in cluster 2 had a shorter PFS and OS compared to cluster 1. In fact, the 5-year PFS estimates were 31% vs 56% (p = 0.0062), whereas the OS probabilities were 66% and 83%, respectively (p = 0.0071). Of note, both hazard ratios for PFS and OS were doubled in pts included in cluster 2, as compared to pts included in cluster 1. Finally, multivariate analyses confirmed that being part of cluster 2 was an independent prognostic factor for both PFS and OS, along with del(17p) and ISS 3. Two alternate Hh-driven subtypes of MM might be identified at diagnosis, which correlated with pts outcomes. Stratification of pts according to their molecular background might help the fine-tuning of future clinical studies. Acknowledgements: FP7 NGS-PTL project, Progetto Regione-Universita 2010/2012 L. Bolondi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Mauro Procacci, Barbara Santacroce, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Serena Rocchi, Katia Mancuso, Beatrice Anna Zannetti, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3189.

Abstract 5022: The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome

Introduction In most human cancer p53 impairment is a driver event, which confers a survival advantage to affected cells. In Multiple Myeloma (MM), the role of p53 clonal aberrations (mainly 17p del) is well recognised, whereas the prognostic relevance of TP53 mutations is less clear, due to the very limited frequency of clonal lesions. Here we aim at characterizing by ultra-deep sequencing (UDS) the TP53 mutational state in both newly diagnosed and relapsed MM pts, to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. Pts and methods A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and ASCT was included in this study. In 29 cases, samples were collected also at relapse(s). DNA was obtained from CD138+ highly purified plasma cells and SNPs array profiled (Affymetrix). TP53 gene was analysed by amplicon-targeted UDS approach (GSJ, 454 Roche). A specific bioinformatics pipeline was set up to discriminate between low frequency TP53 variants and sequencing errors. Results With a median coverage of 1386X, 129 correctly called TP53 variants were detected. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts).. According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with Conclusion The TP53 UDS analysis in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. Acknowledgements: Roche Diagnostics, FP7 NGS-PTL project, Fondazione Berlucchi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Barbara Santacroce, Mauro Procacci, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Beatrice Anna Zannetti, Katia Mancuso, Serena Rocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5022.