1 INTRODUCTION
2.2 Novel drugs in multiple myeloma
2.2.5 Description of novel drugs
2.2.5.6 Other new proteasome inhibitors
2.2.5.6 Other new proteasome inhibitors
Ixazomib is the first oral PI offering the possibility of first all-‐‑oral combination regimen including PIs and IMiDs. In phase 1 trials the once weekly administration proved to be feasible due to the long terminal half-‐‑life (215, 216). A phase 1/2 study including ixazomib, lenalidomide and dexamethasone (IRd) for newly diagnosed (ND) MM gave the basis for a fixed dose of 4 mg for phase 2, with which dose IRd produced ≥ VGPR rate of 58% and PN grade ≥ 3 of only 6% (217). The first analysis of the TOURMALINE-‐‑MM 1 phase 3 study
(NCT01564537) demonstrated a 35% improvement in PFS with the combination of IRd (n=360) compared to placebo-‐‑Rd (n=362) for patients with RRMM (218). The median PFS was 20.6 and 14.7 months, respectively. TTP and response rates were also significantly improved with the IRd combination. The addition of ixazomib combined to Rd increased median PFS without a substantial increase in overall toxicity. In patients with high-‐‑risk cytogenetics, the PFS hazard ratio was 0.543 with IRd vs Rd (hr 0.596 in patients with del(17)), with a median PFS similar to the overall IRd group, indicating that ixazomib may overcome the negative impact of cytogenetic alterations (218). Ixazomib has now proceded to a phase 3 trial for NDMM transplant non-‐‑eligible patients (IRd vs placebo + Rd, NCT01850524) (219) and studies in the maintenance setting are also ongoing (220). Other next generation PIs in phase 2 studies are oprozimib (oral) and marizomib (intravenous) (221).
2.2.5.7 Immune therapies
A potentially important way to improve the treatment efficacy in MM is to include immune therapy: IMiDs, monoclonal antibodies, checkpoint inhibitors, vaccines and chimeric antigen representing T-‐‑cells (222-‐‑225). Anti-‐‑CD20 therapy was a disappointment probably due to low CD20 expression in MM cells, increased complement inhibiting proteins on MM cells, Fc-‐‑γ polymorphism and selective loss of CD20 expression (226). For a target to be feasible in MoAb-‐‑therapy for MM, the target should be expressed on a majority of MM cells or cells involved in angiogenesis or at a higher level than on non-‐‑target cells (227).
Elotuzumab (CS1), daratumumab (CD38), SAR650984 (CD38), MOR202 (CD38), siltuximab (IL-‐‑6), lorvotuzumab (CD56), nBT062 (CD138), dacetuzumab (CD40), lucatumumab (CD40), tabalumab (B-‐‑cell activating factor), milatuzumab (CD74), ulocuplumab (CXCR4) and nivolumab, pembrolizumab and atezolizumab (PD-‐‑1) have all been investigated as MoAbs in MM (9, 223, 224, 225, 227, 228, 229, 231).
Elotuzumab (ELO) is directed against signaling lymphocyte activation molecule F7 (SLAMF7) also called CS1, which is a glycoprotein that is highly expressed in MM cells but also expressed in NK and CD8+ T-‐‑cells (229). As monotherapy it seems to be nearly useless (230) but the synergy of ELO with LEN has been demonstrated in Eloquent-‐‑2 trial with superiority for LEN+ELO+Dex vs LEN+Dex with a PFS of 19.4 months and 14.9 months, respectively (9). Some synergy seems also to be achieved by adding PI to ELO+Dex based on results of comparison of ELO+BZM+Dex vs BZM+Dex, with PFS of 9.7 and 6.9 (231).
Daratumumab has been found to be efficient even as a monotherapy and in combinations and has resulted in significant responses in patients being treated already with BZM, IMiDs and ASCT (7, 8). In a phase 1-‐‑2 study for RRMM, of whom 64% were double-‐‑refractory, the ORR was 36% for daratumumab as monotherapy, and 65% of these who responded did not have progression at 12 months (7). In the SIRIUS trial the OR was 29% in heavily treated and refractory patients (8).
Anti-‐‑PD1 monotherapy has not been as beneficial in MM as in lymphoma (232), but anti-‐‑
PD1 pembrolizumab combined with IMiD has shown efficacy in RRMM (228). The role of checkpoint inhibitors in MM and especially in relation to ASCT will be explored in future studies (233, 234).
2.2.5.8 Epigenetic approach -‐‑ deacetylase inhibitors
DNA methylation and histone modifications are the two main epigenetic mechanisms. In MM, in addition to DNA methylation, histone methylation and acetylation are the major epigenetic changes. As a monotherapy histone deacetylase inhibitors (HDACi) are not effective, but combination of PI plus HDACi has proved to be effective and has a scientific rationale (235). PIs inhibit the degradation of ubiquitinated misfolded proteins, and HDACi interfere with aggresome formation, which contributes to the accumulation of toxic misfolded proteins in MM cells, finally resulting in apoptosis (235). The most important trials in this field have been phase 3 studies with vorinostat + BZM (236), in which addition
of vorinostat had very limited benefit, and the phase 2-‐‑3 Panorama 1-‐‑2 studies, which showed a PFS difference of 11.9 vs 8.1 and 12.5 vs 4.7 months between panobinostat + BZM + Dex vs BZM + Dex, respectively (237, 238). In the subgroup analysis the PFS difference for patients ≥ 2 treatment lines was 12.5 vs 4.7 months (239). In BZM-‐‑refractory patients, 20-‐‑
30% had a benefit when adding panobinostat (239). To avoid the toxicity of these first-‐‑
generation HDAC inhibitors, a selective HDAC6-‐‑specific inhibitor (ricolinostat, ACY-‐‑1215) has been investigated. Ricolinostat has been shown to trigger synergy when combined with CFZ even in BZM-‐‑refractory MM cells (240).
With regard to histone lysine methyltransferase, MMSET is a very interesting. MMSET is upregulated in all cases of t(4;14) myeloma, leading to global genomic alteration of histone patterns and increased expression of oncogenic loci, e.g. NF-‐‑kB (241). This could offer a possibility for targeted therapy (242).
2.2.5.9 Cell cycle and kinase inhibitors
Because cyclin-‐‑D dysregulation is a pathogenetic event in the course of MM, it has been an interest of targeted therapy. The cyclin-‐‑D kinase inhibitor (CDKi) seleciclib was tried to RRMM patients with BZM, but with disappointing results (243). Another CDKi, dinaciclib, has shown efficacy as a monotherapy in RRMM patients (244). The kinesin spindle protein inhibitor, ARRY-‐‑520, has a specific and unique influence on cells because it can arrest cells in mitosis and induce apoptosis, preventing survival signals (235). Due to this unique influence it is now being investigated in phase 1 studies with PIs and IMiDs (245, 246). In the jungle of a growing number of new molecules for MM it would be valuable to find biomarkers to predict the response. For example in regard to filanesib (ARRY-‐‑520) therapy low alfa 1-‐‑acid glycoprotein levels in plasma seems to correlate with better outcome (247).
In the hope of finding similar targeted therapies like imatinib in chronic myeloid leukemia, tyrosine kinase FGFR3 inhibitors, like dovitinib, have been tested in t(4;14) positive MM patients, but again with poor results (248). Insulin-‐‑like growth factor 1-‐‑receptor (IGF-‐‑1R) is a clear theoretical target in cell growth, but very limited clinical benefit has been found when combining IGF-‐‑1R-‐‑ monoclonal antibody with BZM (249).
2.2.5.10 Signal transduction inhibitors
PIs were the first class of drugs focusing on a specific pathway activated in MM cells, the NF-‐‑kB pathway (250-‐‑251). The PI3K/AKT/mTOR pathway is overactive in MM, leading to proliferation, clonal cell expansion, apoptosis inhibition and drug resistance and is probably one of the most important pathways in the pathogenesis of MM (235, 252, 253).
Perifosine and afuresertib act as AKT inhibitors, showing better efficacy when combined with PIs or IMiDs (254, 255, 256). Phospho-‐‑AKT positivity was associated with better PFS (253). Downstream of this pathway is the mTOR complex, which has been targeted by the mTORC1-‐‑inhibitors everolimus and temsirolimus (257-‐‑258). The latter has been combined with BZM in advanced disease (259). The research in general is focused on better understanding the details of inhibiting PI3K/AKT/mTOR pathway in cancer treatment, because single factor inhibition can cause positive feedback by alternative routes, resulting in a new progression after a short primary response (260). mTORC1/C2 and dualPI3K/mTOR inhibitors have therefore been tested (261). Tipifarnib, farnesyltransferase inhibitor (262) and selumetinib (AZD6244), an MEK inhibitor (263), have been tested against the signaling pathway RAS/RAF/MEK/ERK with minimal stabilization activity of disease so far. About 5% of MM patients have been shown to have kinase BRAF mutations (45, 264), and vemurafenib, a BRAF inhibitor, has been proven to have efficacy in BRAF-‐‑
positive patients with advanced, refractory disease (265).
2.2.5.11 Targeting microenvironment
Due to the unique interaction between BMSCs and MM cells, major efforts have been done in recent years to develop agents to interrupt this symbiosis. Hypoxia seems to promote
disease progression, and the hypoxia-‐‑activated prodrug TH-‐‑302 has been combined with BZM, resulting in induction of apoptosis (266, 267). Even though angiogenesis with vascular endothelial growth factor (VEGF) upregulation is thought to be important for the well-‐‑being of MM cells, antiangiogenetic VEGF-‐‑inhibition does not have proven efficacy in MM (268). Plerixafor, a CXCR4 inhibitor, can block the interaction between MM cells and the BM microenvironment, and when used for a chemosensitization with BZM it has showed an ORR of 40% in RRMM (269).
The main recent phase 2-‐‑3 trials for relapsed or relapsed and refractory patients are shown in Table 13.
2.2.6 Novel drugs and allogeneic transplantation
Transplant-‐‑related mortality (TRM) has been 34-‐‑53% in allogeneic stem cell transplantation (allo-‐‑SCT) preceded by myeloablative conditioning in MM (270). To improve the safety, reduced-‐‑intensity conditioning (RIC) with non-‐‑myeloablative regimens was developed.
TRM decreased to 10-‐‑16% with this approach, but in only two out of seven RIC allo-‐‑SCT trials an OS benefit compared to ASCT was noted (270, 271, 272). The design of these studies varies in several aspects in addition to different follow-‐‑up times which hampers the interpretation of the results (270). Both Len and BZM have been evaluated in the post-‐‑allo setting (270). By stimulation of alloreactive T-‐‑ and NK-‐‑cells Len could promote graft-‐‑
versus-‐‑myeloma effect and improve treatment response, but with excess toxicity of T-‐‑cell mediated graft versus host disease (GVHD). For example, in the HOVON-‐‑76 trial 37%
developed acute GVHD and 17% chronic GVHD, which can be life-‐‑threatening (270, 273, 274). BZM has been tested in small trials as post–allo maintenance based on its inhibition of NF-‐‑kB, producing an antimyeloma effect and GVHD suppression (270, 275). A phase 2 allotransplant study including PI in conditioning (bortezomib) and during maintenance (ixazomib vs placebo) for high-‐‑risk patients will be started in a national US study (276). The risk-‐‑benefit ratio of allo-‐‑SCT is considered to be acceptable in selected patients with ultra-‐‑
high-‐‑risk MM, with del(17p), t(4;14) or t(14;16) and 1q21 amplification (>3 copies) and ISS III whose median OS is predicted to be 24 months or less (277). Novel drugs will most probably be combined with different immunological approach in allo-‐‑SCT in future (270, 279).
2.3 RESPONSE ASSESSMENT IN MULTIPLE MYELOMA 2.3.1 General
The importance of attaining CR with the initial myeloma treatment has become apparent (278, 279). Accordingly, the development of new effective treatment strategies has raised the question of how to compare the efficacy of different therapies more precisely. An immunofixation electrophoresis (IFE) negative CR was incorporated into the remission criteria first in the allogeneic transplantation setting (280). Following the introduction of high-‐‑dose treatment supported by ASCT the new consensus criteria for evaluating disease responses were published, including an IFE-‐‑negative sustained CR (281). Near complete response (nCR) was used for response between partial response (PR) and CR when electrophoresis was normal but immunifixation was still positive (278). In 2006 the International Myeloma Working Group (IMWG) published the consensus criteria for assessing the response in MM. Categories of stringent CR (sCR) and very good partial remission (VGPR) were established, and nCR was included in VGPR (282). The serum FLC (sFLC) assay for response evaluation of oligo-‐‑ or non-‐‑secretory myeloma was also included (282). Kyle & Rajkumar updated ISS and high-‐‑risk criteria in 2009 (283). IMWG updated the response and progression criteria in 2011 (Table 14 and 15), and the terms stringent CR, immunophenotypic CR and molecular CR (MolR) have been suggested for uniform reporting of clinical trials (284).
IFE, sFLC and MFC have been compared in one trial where immunophenotypic remission predicted a longer PFS and TTP than conventional CR or sCR (285). The impact of normalization of sFLC ratio in CR patients is controversial due to the low specificity and the presence of aberrant oligoclonal bands after therapy (286). In addition, normalization of sFLC ratio has been showed in spite of a positive IFE (287). Molecular CR is defined as a CR plus negative ASO-‐‑PCR with sensitivity of 10-‐‑5 (284). For an immunophenotypic remission at least 1 x 106 BM cells should be analyzed using at least 4-‐‑color MFC showing no aberrant clonal plasma cells (284). The sensitivity of the analysis can be improved, even if panels vary, by analyzing as many BM cells as possible even up to 2-‐‑5 x 106 and using ≥ 8 color
MRD can be assessed using immunophenotypic MFC and molecular techniques (PCR based assays and sequencing of IgH locus) from bone marrow samples, or extramedullary molecular or magnetic resolution imaging outside the BM (11, 97, 98, 288). Tables 14 and 15 show the IMWG response criteria for MM.
Table 14. Response criteria for multiple myeloma (284) Stringent complete response (sCR)
Complete response as described in CR and Normal free light chain ratio (FLC) ratio and
Absence of clonal plasma cells by immunohistochemistry or 2- to 4-color flow cytometry Complete response (CR)
Negative immunofixation of serum and urine and Disappearance of any soft tissue plasmacytomas and
< 5% plasma cells in bone marrow
If only measurable disease is by serum FCL levels: CR indicates a normal FCL ratio of 0.26 to 1.65 in addition to CR criteria listed above
Very good partial response (VGPR)
Serum and urine monoclonal protein (M-component) detectable by immunofixation but not on by electrophoresis or
90% or greater reduction in serum M-component plus urine M-component level < 100 mg per 24 hours
If only measurable disease is by serum FCL levels: VGPR indicates > 90% decrease in the difference between involved and uninvolved FCL levels
Partial response (PR)
≥50% reduction of serum M-protein and reduction in 24-hour urinary M-component by ≥90%
or to < 200 mg/24 h
If the serum and urine M-protein are not measurable, a ≥50% decrease in the difference between involved and uninvolved FLC levels
If the serum and urine M-protein are not measurable, and S-FLC is also unmeasurable, ≥50%
reduction in plasma cells provided bone marrow plasma cell percentage was ≥30%
In addition to the above, if present at baseline a ≥50% reduction in the size of soft plasmacytomas
Stable disease (SD)
Not meeting criteria for CR, VGPR, PR or progressive disease Progressive disease (PD)
Increase of 25% from lowest response value in any of the following - Serum M-component; absolute increase must be ≥ 0.5 g/dl and /or - Urine M-component; absolute increase must be ≥200 mg/24 h and/or
In patients without measurable S/U-M-prot difference between involved and uninvolved FCL levels (absolute increase > 10mg/dl)
Bone marrow PC % (must be ≥ 10%, only in patients without measurable M-component or FLC)
New bone lesions or plasmacytomas or growth of them Development of hypercalcemia
Table 15. Additional response criteria for multiple myeloma (284)
Mimimal response in patients with relapsed refractory myeloma adopted from the EBMT criteria (284)
≥ 25% but ≤ 49% reduction of S-M-protein and reduction in 24h U-M-protein by 50%-89%
In addition to the above, if present at baseline, 25%-49% reduction in the size of soft tissue plasmacytomas is also required
No increase in size or number of lytic bone lesions (development of compression fracture does not exclude response)
Immunophenotypic remission
Stringent CR and absence of phenotypic aberrant PCs (clonal) in BM with a minimum of 1 million total BM cells analyzed by MFC (with > 4 colors)
Molecular CR
CR and negative ASO-PCR with sensitivity 10-5
In addition to the above mentioned criteria refractory myeloma is a status of myeloma that is nonresponsive during primary or salvage treatment, or is progressing within 60 days from the last therapy. Nonresponding myeloma is the phase of MM with failure to reach minimal response (MR) or progression during therapy. Two different categories are included in refractory MM, namely primary refractory myeloma and relapsed and refractory myeloma (RRMM). RRMM is nonresponsive during salvage therapy, or is progressive within 60 days from the last therapy in patients who have reached at least MR at some time point before this progression. Primary refractory MM means that the patient never achieved even MR and is either nonresponsive or nonprogressive or the patient is not only nonresponsive, but is progressing. Relapsed MM denotes MM that initially responded to treatment, but is progressing and is in need of salvage treatment. This needs to be separated from primary refractory MM and RRMM. Especially in clinical trials it should be described to which drug category the patients have relapsed or are refractory, in addition to use of stratification factors like cytogenetic aberrations and disease stage (284).
2.3.2 Assessment of minimal residual disease (MRD) in myeloma patients 2.3.2.1 Multiparameter flow cytometry (MFC)
The first goal of the EuroFlow Consortium was to establish a diagnostic panel for PC disease, and it has been thereafter updated for MFC-‐‑MRD assessment (92, 93, 94). If the goal is to assess MRD negative status, the minimum number of cellular events is > 500 000.
Consensus guidelines recommend 2 x 106 cells as an acceptable minimum for MRD and as much as 5 x 106 cells for high-‐‑sensitivity MRD (93, 94). A novel MFC method is being planned that will use 8-‐‑10 (-‐‑12) polychromatic cytometry with novel software enabling more precise discrimination of clonal and normal PCs. Development of reference library of normal and tumor cells and a (semi) automated flow-‐‑MRD monitoring are also being planned (12).
One of the goals of EuroFlow Consortium was to develop the MFC method so that with very distinguished panel it would be possible to identify MRD even without the primary sample from each patient as a reference. Using the automated computer model-‐‑based, novel MFC analysis (principal component analysis, PCA) Paiva et al. were able to recognize a group of newly diagnosed MM with a MGUS-‐‑like profile with long-‐‑term OS independent of CR response (289). PC antigen expressions have also been investigated for outcome prediction. Mateo et al. observed that CD28+ PCs associated with t(14;16) and del(17p), and CD117− PCs with t(4;14) and del 13q and CD28+/CD117-‐‑ immunophenotype was a poor prognostic marker for PFS and OS (290). CD56 expression of MM cells indicates BM stroma
extramedullary manifestations (291). Loss of CD56 expression in the course of myeloma can also predict the risk of developing plasma cell leukemia (292).
Figure 2. Example of ten color multiparameter flow cytometry for detection of minimal residual disease in multiple myeloma. The antibody panel was following: CD38 FITC /cylambda PE /CD56 ECD /CD138 PC5.5 /CD19 PC7 /cykappa APC /CD200 APC R700 /CD81 APC H7 /CD27 BV421 /CD45 KO. Analysis was made with Navios (Beckman-Coulter) flow cytometry and Infinicyt (Cytognos) soft-ware.
FITC= fluorescein isothyocyanate, PE= phycoerythrin, ECD= PE-Texas Red, PC5.5=PE Cyanin5.5, PC7=PE Cyanin7, APC= allocyanin, BV421=Brilliant Violet 421, KO=Krome Orange.
CD38 clone L38 from Cytognos, CD56 clone NHK1, CD138 clone B-A38, CD19 clone J3-119, CD45 clone J.33 from Beckman Coulter, CD200 clone OX104, CD81 clone JS-81 and CD27 clone M-T271 from Beckton Dickinson, lambda and kappa, polyclonal from Dako.
The first positive studies regarding MFC-‐‑MRD assessment in MM encouraged the Spanish and UK groups to include their 4-‐‑ and 6-‐‑ color MFC-‐‑MRD panels in clinical trials.
In both the PETHEMA/GEM2000 and MRC Myeloma IX study the achievement of immunophenotypic remission after ASCT was predictive to superior PFS and OS (293, 294, 295, 296). These studies showed the impact of immunophenotypic remission for patients in conventional CR as well, but the achievement of MRD-‐‑negativity in high-‐‑risk patients was most predictive of the more favorable outcome and sustained CR (295, 296, 297). They also demonstrated that MFC-‐‑MRD negativity independent of serological PR or CR might predict similar or even better outcome than CR status with MRD-‐‑positivity (285).
Including two novel agents in the treatment regimen Roussel et al. were able to achieve MFC-‐‑MRD negativity in up to 68% of patients using the 7-‐‑color method in MRD assessment (150). This response correlated again with longer PFS. The threshold of MRD negativity was limited to 0.01% in the first studies (293-‐‑296, 298, Table 16) due to technical reasons compared to the sensitivity capabilities of 10-‐‑5 with novel MFC methods. In addition, to MFC-‐‑MRD negativity the MRD level as a continuous variable seems to be predictive for OS. In MRC Myeloma IX trial there was a significant improvement in OS for
each log depletion in MRD level (299). Studies of using MFC in MM patients are summarized in Table 16.
Table 16. Main studies of MFC-MRD detection and outcome of MFC – and MFC + patients
Reference Treatment No. Method/Limit
of detection Appl./
Appl., applicability; MRD, minimal residual disease; MFC, multiparameter flow cytometry; CT, chemotherapy; NA, not available; mo, months; y, years; VMP, bortezomib, melphalan,
Appl., applicability; MRD, minimal residual disease; MFC, multiparameter flow cytometry; CT, chemotherapy; NA, not available; mo, months; y, years; VMP, bortezomib, melphalan,