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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 signaturewas 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.

The prognosis for patients multiple myeloma (MM) has improved substantially over the past decade with the development of new, more effective chemotherapeutic agents and regimens that possess a high level of anti-tumor activity. In spite of this important progress, however, nearly all MM patients ultimately relapse, even those who experience a complete response to initial therapy. Management of relapsed MM thus represents a vital aspect of the overall care for patients with MM and a critical area of ongoing scientific and clinical research. This comprehensive manuscript from the International Myeloma Working Group provides detailed recommendations on management of relapsed disease, with sections dedicated to diagnostic evaluation, determinants of therapy, and general approach to patients with specific disease characteristics. In addition, the manuscript provides a summary of evidence from clinical trials that have significantly impacted the field, including those evaluating conventional dose therapies, as well as both autologous and allogeneic stem cell transplantation. Specific recommendations are offered for management of first and second relapse, relapsed and refractory disease, and both autologous and allogeneic transplant. Finally, perspective is provided regarding new agents and promising directions in management of relapsed MM.

Identification of patient sub-groups with smoldering multiple myeloma (SMM) at high risk of progression to active disease (MM) is an important goal. 18F-FDG PET/CT (positron emission tomography (PET) integrated with computed tomography (PET/CT) using glucose labelled with the positron-emitting radionuclide 18F) allows for assessing early skeletal involvement. Identification of osteolytic lesions by this technique has recently been incorporated into the updated International Myeloma Working Group criteria for MM diagnosis. However, no data are available regarding the impact of focal lesions (FLs) without underlying osteolysis on time to progression (TTP) to MM. We hence prospectively studied a cohort of 120 SMM patients with PET/CT. PET/CT was positive in 16% of patients (1 FL: 8, 2 FLs: 3, >3 FLs: 6, diffuse bone marrow involvement: 2). With a median follow-up of 2.2 years, 38% of patients progressed to MM, in a median time of 4 years, including 21% with skeletal involvement. The risk of progression of those with positive PET/CT was 3.00 (95% confidence interval 1.58–5.69, P=0.001), with a median TTP of 1.1 versus 4.5 years for PET/CT-negative patients. The probability of progression within 2 years was 58% for positive versus 33% for negative patients. In conclusion, PET/CT positivity significantly increased the risk of progression of SMM to MM. PET/CT could become a new tool to define high-risk SMM.

The role of high-dose therapy and autologous stem-cell transplantation (ASCT) in the treatment of multiple myeloma (MM) has continued to evolve in recent years. The novel agents thalidomide, bortezomib, and lenalidomide have been successfully incorporated into induction therapy in preparation for ASCT and are currently being investigated also as post-ASCT consolidation and maintenance therapy. Consolidation treatment is generally short term and aims to increase the frequency and depth of response obtained with the previous treatment phases, including novel agent-based induction therapy and ASCT. This review will focus on recent trials of novel agents as post-ASCT consolidation therapy, offering an overview of pros and cons of this new treatment strategy in the ASCT sequence for MM patients.

Thalidomide is effective as a first-line therapy for the treatment of multiple myeloma (MM), but its use is limited by peripheral neurotoxicity.

In recent years, treatment paradigm of targeting the “soil” bone marrow (BM) microenvironment as a means of interfering with the growth of the MM “seed” has provided the rationale for investigational clinical trials of novel agents combined with old drugs in an attempt to maximize tumour response. Thalidomide, which represents an effective treatment strategy for relapsed/refractory MM, actually represents a standard of care also for newly diagnosed MM patients. We have recently demonstrated that thali-dex combination as front-line therapy in preparation for autologous transplantation is superior to VAD in terms of increased rate of response (≥ partial response: 76%) and magnitude of tumour reduction. In particular, the probability to attain at least a very good partial response (VGPR) was 19%, including 13% of patients in complete response (CR) or near CR (nCR). In the present study we adopted a GEP strategy in an attempt to identify a signature able to predict the probability to attain ≥ nCR to combined thali-dex as upfront therapy for patients with newly diagnosed MM. CD138+ samples obtained at diagnosis from 32 patients enrolled in the “Bologna 2002” clinical trial were used throughout the study; all patients were evaluable for response to thali-dex in preparation for autologous transplantation. GEP was performed using the Affymetrix HG133 Plus microarray platform. The Affymetrix output (CEL files) was imported into Genespring 7.3 (Agilent technologies) microarray analysis software, where data files were normalized across chips using GCRMA and to the 50th percentile, followed by per gene normalization to median. Genes differently expressed in subgroups of patients were selected by an ANOVA analysis. Criteria of response were those established by Bladè et al, with the addition of a VGPR and nCR categories. Overall, six of the 32 patients (19%) obtained at least a nCR to thali-dex, whereas the remaining 26 patients either achieved a partial response or did not respond. We identified a gene signature of 162 genes, able to significantly distinguish patients with ≥ nCR from the others (p=0.05). We then adopted a Nearest-Neighbours (NN) classifier (using 3 first neighbours), with a “Leave-one-out Cross Validation” procedure, to identify a list of ten genes able to predict the ≥ nCR in our series of patients (see table). Of interest, the gene list encompasses CCND2, one of the most important cell cycle regulator known to be involved in tumour progression in MM patients and the anti-apoptotic gene CFLAR, both down-regulated in nCR patients. These results could be the first step to adopt microfluidic cards, in an attempt to select at diagnosis patients who will respond very favourably to a particular treatment strategy. Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell’Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo. gene ID gene name chromosomal location 225792_at HOOK1 1p32.1 236223_s_at unknown 1q22 225282_at SMAP1L 1p35.3-p34.1 239629_at CFLAR 2q33-q34 227027_at GFPT1 2p13 226886_at GFPT1 2p13 205848_at GAS2 11p14.3-p15.2 200951_s_at CCND2 12p13 200628_s_at TSPAN4 14q32.31 242121_at unknown unknown

In MM tandem duplication and jumping translocations of the q21 band of chromosome (chr.) 1 are acquired during progression of the disease and can lead to chr.1q amplification. In several studies, chr. 1 amplification has been linked to poor prognosis after both conventional and high-dose chemotherapy. Recently, a subset of genes mapping on 1q21 band has been identified, whose increased expression may be due to increased DNA copy number. Among these genes, CKS1B has been one of the most significantly upregulated. CKS1B regulates SCF skp2 mediated ubiquitination and proteolysis of the cyclin dependent kinase inhibitor p27Kip1, whose low expression has been reported to be an independent adverse prognostic factor in MM patients. Aim of the present study was to investigate the relationship between CKS1B expression and response to primary therapy with thali-dex in a large series of patients with newly diagnosed MM. Secondary endpoint was to explore the relationship between CKS1B expression and del(13), as assessed by FISH analysis, and t(4;14), as evaluated by an RT-PCR assay designed to detect the presence of IgH/MMSET fusion gene. A total of 132 patients were analyzed. The presence of t(4;14) and CKS1B expression were investigated in all patients, while del(13) was studied in 129/132 patients. CKS1B expression was evaluated by Real-time RT-PCR. CKS1B values were separated in four different quartiles, with expression levels increasing progressively from quartile 1 to 4. Response to therapy was evaluated according to the criteria proposed by Bladè et al. The Fisher test and the Mann-Whitney test were applied for statistical analysis. On an intent-to-treat basis, the overall probability to respond (≥ partial response, PR) to up-front thali-dex therapy was 71%, while 38 patients (29%) either did not respond (NR) or progressed. Median CKS1B expression value was significantly higher in NR in comparison with patients who attained at least a PR: 1.42 (range 0.15–52.35) vs. 0.89 (range 0–11.88), respectively (p=0.01). In particular, the proportion of NR patients in the CKS1B expression quartile 4 was significantly higher as opposed to the frequency of NR in the CKS1B expression quartiles 1 to 3 (45.5% vs. 23.2%, respectively; p= 0.02). CKS1B over expression did not correlate with the presence of t(4;14) or del(13). Only 6 patients harbouring t(4;14) fell into the CKS1B expression quartile 4, as opposed to 32 patients included into the CKS1B expression quartiles 1–3 (18.2% vs. 32.3%; p, not significant). Similarly, the frequency of del(13) was comparable in the CKS1B expression quartile 4 and in the CKS1B expression quartiles 1–3 (34.4% vs. 44.3%; p, not significant). Likewise, only 2 patients carrying both t(4;14) and del(13) fell into the CKS1B expression quartile 4, as opposed to 15 patients who fell into the CKS1B expression quartiles 1–3 (6.5% vs. 15.3%; p, not significant). In conclusion, in patients with newly diagnosed MM, CKS1B over expression at baseline predicts for a significantly lower probability of response to primary remission induction therapy with thali-dex. Poor response to thali-dex conferred by CKS1B over expression is independent from the presence of t(4;14) and/or del(13). Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell’Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo.

In MM tandem duplication and jumping translocations of the q21 band of chromosome (chr.) 1 are acquired during progression of the disease and can lead to chr.1q amplification. In several studies, chr. 1 amplification has been linked to poor prognosis after both conventional and high-dose chemotherapy. Recently, a subset of genes mapping on 1q21 band has been identified, whose increased expression may be due to increased DNA copy number. Among these genes, CKS1Bhas been one of the most significantly upregulated. CKS1B regulates SCF skp2 mediated ubiquitination and proteolysis of the cyclin dependent kinase inhibitor p27Kip1, whose low expression has been reported to be an independent adverse prognostic factor in MM patients. Aim of the present study was to investigate the relationship between CKS1Bexpression and response to primary therapy with thali-dex in a large series of patients with newly diagnosed MM. Secondary endpoint was to explore the relationship between CKS1Bexpression and del(13), as assessed by FISH analysis, and t(4;14), as evaluated by an RT-PCR assay designed to detect the presence of IgH/MMSET fusion gene. A total of 132 patients were analyzed. The presence of t(4;14) and CKS1Bexpression were investigated in all patients, while del(13) was studied in 129/132 patients. CKS1Bexpression was evaluated by Real-time RT-PCR. CKS1Bvalues were separated in four different quartiles, with expression levels increasing progressively from quartile 1 to 4. Response to therapy was evaluated according to the criteria proposed by Bladè et al. The Fisher test and the Mann-Whitney test were applied for statistical analysis. On an intent-to-treat basis, the overall probability to respond (≥ partial response, PR) to up-front thali-dex therapy was 71%, while 38 patients (29%) either did not respond (NR) or progressed. Median CKS1Bexpression value was significantly higher in NR in comparison with patients who attained at least a PR: 1.42 (range 0.15–52.35) vs. 0.89 (range 0–11.88), respectively (p=0.01). In particular, the proportion of NR patients in the CKS1Bexpression quartile 4 was significantly higher as opposed to the frequency of NR in the CKS1Bexpression quartiles 1 to 3 (45.5% vs. 23.2%, respectively; p= 0.02). CKS1Bover expression did not correlate with the presence of t(4;14) or del(13). Only 6 patients harbouring t(4;14) fell into the CKS1Bexpression quartile 4, as opposed to 32 patients included into the CKS1Bexpression quartiles 1–3 (18.2% vs. 32.3%; p, not significant). Similarly, the frequency of del(13) was comparable in the CKS1Bexpression quartile 4 and in the CKS1Bexpression quartiles 1–3 (34.4% vs. 44.3%; p, not significant). Likewise, only 2 patients carrying both t(4;14) and del(13) fell into the CKS1Bexpression quartile 4, as opposed to 15 patients who fell into the CKS1Bexpression quartiles 1–3 (6.5% vs. 15.3%; p, not significant). In conclusion, in patients with newly diagnosed MM, CKS1Bover expression at baseline predicts for a significantly lower probability of response to primary remission induction therapy with thali-dex. Poor response to thali-dex conferred by CKS1Bover expression is independent from the presence of t(4;14) and/or del(13). Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell'Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo.

In recent years, treatment paradigm of targeting the “soil” bone marrow (BM) microenvironment as a means of interfering with the growth of the MM “seed” has provided the rationale for investigational clinical trials of novel agents combined with old drugs in an attempt to maximize tumour response. Thalidomide, which represents an effective treatment strategy for relapsed/refractory MM, actually represents a standard of care also for newly diagnosed MM patients. We have recently demonstrated that thali-dex combination as front-line therapy in preparation for autologous transplantation is superior to VAD in terms of increased rate of response (≥ partial response: 76%) and magnitude of tumour reduction. In particular, the probability to attain at least a very good partial response (VGPR) was 19%, including 13% of patients in complete response (CR) or near CR (nCR). In the present study we adopted a GEP strategy in an attempt to identify a signature able to predict the probability to attain ≥ nCR to combined thali-dex as upfront therapy for patients with newly diagnosed MM. CD138+ samples obtained at diagnosis from 32 patients enrolled in the “Bologna 2002” clinical trial were used throughout the study; all patients were evaluable for response to thali-dex in preparation for autologous transplantation. GEP was performed using the Affymetrix HG133 Plus microarray platform. The Affymetrix output (CEL files) was imported into Genespring 7.3 (Agilent technologies) microarray analysis software, where data files were normalized across chips using GCRMA and to the 50thpercentile, followed by per gene normalization to median. Genes differently expressed in subgroups of patients were selected by an ANOVA analysis. Criteria of response were those established by Bladè et al, with the addition of a VGPR and nCR categories. Overall, six of the 32 patients (19%) obtained at least a nCR to thali-dex, whereas the remaining 26 patients either achieved a partial response or did not respond. We identified a gene signature of 162 genes, able to significantly distinguish patients with ≥ nCR from the others (p=0.05). We then adopted a Nearest-Neighbours (NN) classifier (using 3 first neighbours), with a “Leave-one-out Cross Validation” procedure, to identify a list of ten genes able to predict the ≥ nCR in our series of patients (see table). Of interest, the gene list encompasses CCND2, one of the most important cell cycle regulator known to be involved in tumour progression in MM patients and the anti-apoptotic gene CFLAR,both down-regulated in nCR patients. These results could be the first step to adopt microfluidic cards, in an attempt to select at diagnosis patients who will respond very favourably to a particular treatment strategy. Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell'Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo.

Insulin-like growth factors 1 (IGF1) plays an important role in regulating cell proliferation, differentiation, apoptosis, and transformation. IGF1 acts by binding the type 1 IGF receptor (IGF-1R), thus activating its tyrosine kinase activity. Recent studies have shown that IGF1 is an important survival and growth factor in multiple myeloma (MM). In this study we evaluated the expression of IGF1 and IGF1-R in a series of 49 newly diagnosed MM patients (pts) primarily treated with thalidomide and dexamethasone. The aim of the study was to correlate the expression of these genes with presenting features of MM pts and to evaluate their relationship with response to therapy. We isolated the CD138+ cell fraction from bone marrow (BM) samples; IGF1 and IGF1-R expression was evaluated by Real-time RT PCR in both CD138+ and CD138- cells, considering a pool of donors as calibrator (expression value=1). We found that neoplastic CD138+ and CD138- cells expressed widely high levels of IGF1 (median 145.01 and 3.16, range 0.13–1089.92 and 0.02–103.25, respectively), and low levels of IGF1-R (median 0.76 and 2.27, range 0.04–9.38 and 0.21–13.55, respectively). As expected, expression data for both genes, in both cell populations resulted very scattered; in fact no differences could be highlighted in the expression of IGF1 and IGF1-R genes, with respect neither to presenting clinical features, nor to the presence of two of the most frequent MM chromosomal alterations (t(4;14) and del(13)). According to response to induction therapy, pts were divided in two subgroups, including 35 responsive and 14 non responsive pts, respectively. Again, overall no relationship was pointed out between the expression of IGF1 and IGF1-R and response to therapy. IGF1 is known to regulate IGF1-R at transcriptional level, down-regulating its expression; this mechanism may be autocrine or paracrine. We thus hypothesized that the unexpectedly low IGF1-R expression levels might be the consequence of a negative-feedback regulation mechanism, induced by an IGF1autocrine effect. Nevertheless, we were not able to highlight a significant inverse correlation between the expression values of IGF and IGF1-R in any of the cell fraction analyzed. On the contrary, among responsive pts, a highly significant inverse correlation was highlighted between IGF1 expressed by the CD138+ and IGF1-R expressed by the CD138− cells (p= 0.001), thus suggesting a possible paracrine effect of IGF1 produced by plasma cells exerted on CD138− cells; interestingly, the same effect could not be highlighted in pts non responding to induction therapy. In conclusion, our preliminary data confirmed the possible involvement of IGF1/IGF1R pathway in MM pathogenesis; we suggested the existence of a paracrine effect of IGF1 produced by CD138+ cells, thus emphasizing the importance of the dialog between plasma cells and microenvironment in MM. Moreover, as this mechanism seemed to act only in responding pts, the ability to efficiently regulate IGF1-R expression may have an important prognostic value. The study of post transduction modifications of the IGF1-R will thus be needed, in order to get more insight into the relationship between the IGF and IGF1-R expressions and the receptor activation.

Insulin-like growth factors 1 (IGF1) plays an important role in regulating cell proliferation, differentiation, apoptosis, and transformation. IGF1 acts by binding the type 1 IGF receptor (IGF-1R), thus activating its tyrosine kinase activity. Recent studies have shown that IGF1 is an important survival and growth factor in multiple myeloma (MM).