Immunomodulatory drugs in AL amyloidosis

Accepted Manuscript Title: Immunomodulatory drugs in AL amyloidosis Author: T. Jelinek Z. Kufova R. Hajek PII: DOI: Reference:

S1040-8428(16)30004-X http://dx.doi.org/doi:10.1016/j.critrevonc.2016.01.004 ONCH 2113

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Critical Reviews in Oncology/Hematology

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25-10-2014 20-11-2015 12-1-2016

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Immunomodulatory drugs in AL amyloidosis

Authors: T. Jelinek1, 2, Z. Kufova2, R. Hajek1, 2

1

Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic

2

Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic

CORRESPONDING AUTHOR: Prof. Roman Hajek, MD, PhD Department of Haematooncology University Hospital Ostrava 17. listopadu 1790, 708 52 Ostrava Czech Republic Phone: +420 597 37 2092 Fax: +420 597 37 2092 Email: [email protected]

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Abstract Immunoglobulin light chain amyloidosis (AL amyloidosis) is indeed a rare plasma cell disorder, yet the most common of the systemic amyloidoses. The choice of adequate treatment modality is complicated and depends dominantly on the risk stratification of these fragile patients. Immunomodulatory drugs (IMiDs) are currently used in newly diagnosed patients as well as in salvage therapy in relapsed/refractory patients. IMiDs have a pleiotropic effect on malignant cells and the exact mechanism of their action has been described recently. Thalidomide is the most ancient representative, effective but toxic. Lenalidomide seems to be more effective, nevertheless the toxicity remains high, especially in patients with renal insufficiency. Pomalidomide is the newest IMiD used in this indication with a good balance between efficacy and tolerable toxicity and represents the most promising compound. This review is focused on the evaluation of all three representatives of IMiDs and their roles in the treatment of this malignant disorder.

Keywords: AL amyloidosis; immunomodulatory drugs; thalidomide; lenalidomide; pomalidomide

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1. Introduction Immunoglobulin light chain amyloidosis (AL amyloidosis, AL) is a low tumor burden plasma cell disorder characterized by the presence of a small indolent clone of plasmocytes in bone marrow (BM) synthesizing misfolded light chains. Instead of conforming to the α-helical configuration of most proteins, these amyloidogenous light chains form a ß-pleated sheet responsible for formatting insoluble fibrils causing damage to vital organs (Bhat et al., 2010). Although AL is a very rare disease, it is the most common systemic amyloidosis with an incidence of 10 patients per million cases per year, resulting in approximately 5000 new patients/year in the European Union (Merlini and Palladini, 2013). Due to the nature of the disease, patients usually present with multi-organ dysfunction (involving the cardiac, renal, gastrointestinal, peripheral and autonomic nervous system), which is essential to their prognosis. The presence and extent of heart involvement is the most important prognostic determinant, and cardiac dysfunction may be best assessed by the cardiac biomarkers Nterminal pro-natriuretic peptide type-B (NT-proBNP), brain natriuretic peptide (BNP) and troponins (Gatt and Palladini, 2013). A staging system based on these biomarkers was revised by Kumar (S. Kumar et al., 2012) and allows to discriminate between low-risk, intermediate-risk and high-risk patients, who have significantly different prognoses. According to this Mayo Clinic staging system, the physicians should choose the appropriate treatment approach based on the fragility of patients. The goal of the treatment in AL amyloidosis is the rapid elimination of toxic free light chains and the achievement of complete hematological remission (Palladini et al., 2012a), which is necessary for amelioration of organ function (Merlini and Palladini, 2008). Achieving a maximal hematological response is a crucial factor and only under these conditions the organ treatment response can be expected. Reaching organ treatment response may last a long time (1-2 years) and is especially important in patients with advanced cardiac involvement (Wechalekar et al., 2013). Disease response and progression criteria are summarized in Table 1 (Comenzo et al., 2012; Gertz et al., 2005; Palladini et al., 2012a). The conventional treatment of AL amyloidosis is based on a combination of corticosteroids (prednisone, dexamethasone) and alkylators (melphalan, cyclophosphamide). This combination used to be a standard treatment option for a long time, first used in 1972 (Jones et al., 1972). The effects of these two groups of compounds are mutually potentiated. The oral combination of melphalan and prednisone is well tolerated in patients with AL, but for a better treatment response, dexamethasone is usually used instead of prednisone (Dhodapkar et al., 1997; Kyle et al., 1997; Skinner et al., 1996). In the clinical trial combining oral melphalan and high-dose dexamethasone in patients ineligible for autologous stem cell transplantation (ASCT), median progression-free and overall survival were 3.8 and 5.1 years, respectively, with an estimated overall response rate (ORR) of 67% including complete remission (CR) in 1/3 of patients. Oral combination of melphalan and high dose dexamethasone has become widely accepted as a first line treatment option for patients with AL amyloidosis ineligible for 3

ASCT (Palladini et al., 2007, 2004). Interestingly, the same Italian group recently reported the results of a large retrospective study showing that the treatment outcome of this combination is dependent on the fraction of patients with advanced cardiac amyloidosis, respectively on the doses of dexamethasone they are able to tolerate. Hematological response was achieved in 76% (CR in 31%) of patients who were fit enough to receive melphalan with full-dose dexamethasone, while only 51% (CR 12%) of patients responded to melphalan with attenuated dexamethasone (Giovanni Palladini et al., 2014). Autologous stem cell transplantation (ASCT) was described for the first time in 1998 as an effective treatment modality in AL amyloidosis (Comenzo et al., 1998). The most commonly used myeloablative regimen is melphalan 200 mg/m2, the same as in multiple myeloma patients, but with frequent dose reductions with consideration to renal or cardiac insufficiency. Recently, in the XIVth International Symposium on Amyloidosis, a cohort of 148 patients who had undergone risk adapted ASCT and had received melphalan 100, 140 and 200 mg/m2 in 14%, 52% and 34% respectively was presented. Interestingly, by multivariate analysis, the melphalan dose had no impact on overall survival () (p = 0.25) (Landau et al., 2014). Complete remissions can be expected in 30 – 40% of cases using this therapeutical approach (Dispenzieri et al., 2013; Gertz et al., 2004; Sanchorawala et al., 2007a; Skinner et al., 2004). Only one prospective multicentric randomized clinical trial comparing ASCT with oral melphalan + dexamethasone was conducted, but the results did not support the superiority of ASCT. Conversely, median OS was only 22.2 months in the ASCT group vs. 56.9 months in the melphalan + dexamethasone group(p = 0.04) (Jaccard et al., 2007). However, these results must be interpreted very carefully because of high peritransplant mortality (24%) related to the inappropriate choice of transplant eligible candidates and some protocol difficulties in the ASCT group. In 2013 two large retrospective studies supporting the use of ASCT in suitable patients with AL amyloidosis were published. An European Group for Blood and Marrow Transplantation (EBMT) analysis of 1315 patients with AL showed time progress in treatment results and 1 year OS reached 91% in the period between 2009 – 2010 in comparison to 65% in the period between 1997 – 1999 (Hegenbart et al., 2013). Another analysis from The Amyloidosis Center at Boston University with 593 patients with AL treated with ASCT during last 19 years presented remarkable long – term survival results with a CR rate of 40%, median OS 6.7 years and 25% of patients living longer than 19 years after ASCT (Sanchorawala et al., 2014a). In conclusion, it is possible to state that with appropriate patient selection and a risk-adapted treatment approach, ASCT is a safe and effective modality for approximately 30% of patients with AL. The reported 10 year survival after ASCT is about 43% (Cordes et al., 2012). The already mentioned adequate selection of patients with limited cardiac involvement is indeed the most important factor for the safety of ASCT. Patients who have troponin T > 0.06 ng/ml, NT-proBNP > 5000 pg/ml and systolic blood pressure < 100 mm Hg at

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diagnosis should not be considered for high-dose chemotherapy (Gatt and Palladini, 2013; Girnius et al., 2014). Proteasome inhibitors (PIs) represent a very effective group of drugs used in the treatment of AL amyloidosis. The majority of clinical studies were conducted with bortezomib. Although there are no available results of any prospective randomized clinical trials, data coming from ongoing clinical trials phase II are very encouraging. In patients with monotherapy of bortezomib, 67 – 69% achieved hematological response and 24 – 38% achieved CR (Reece et al., 2011). The combination of bortezomib and dexamethasone achieved a hematological response in 80 – 94% of cases (Kastritis et al., 2007). Incorporating cyclophosphamide with bortezomib and dexamethasone (CVD) proved clinical efficacy with an ORRORR in 81% (CR 42%) of patients, 2 year progression-free survival (PFS) and OS were 67% and 98%, respectively (Venner et al., 2012). These results were confirmed in many other clinical trials. Rapid treatment response was also observed (Gatt and Palladini, 2013). Recently, a large retrospective matched control case study of 184 newly diagnosed patients with AL was published. The authors compared the outcome of 87 patients treated with bortezomib plus melphalan and dexamethasone (BMDex) with that of 87 controls treated with MDex. A higher proportion of complete responses were observed in the BMDex cohort (42% vs. 19%), and this resulted in a higher overall hematological response rate (69% vs. 51%), but it did not result in the survival improvement in the overall population. However, a significant survival advantage for BMDex was observed in patients without severe (New York Heart Association class III or IV) heart failure and with NT-proBNP < 8500 ng/l. Patients treated with full-dose dexamethasone had similar response rates and survival whether they received bortezomib or not. So, intermediate-risk patients who are not fit enough to receive high-dose dexamethasone are likely to have the greatest advantage from the addition of bortezomib to MDex (G. Palladini et al., 2014). In conclusion, no phase III or retrospective matched analysis indicates a clear significant benefit in the terms of OS/PFS compared to standard treatment regimen and there is still no certainty about the role of upfront bortezomib in the overall patient population with AL amyloidosis (Dispenzieri, 2014). This question should be answered in the near future when the results of phase III randomized multicenter clinical trial EMN03 comparing MDex versus BMDex (bortezomib, melphalan, dexamethasone) will be published. Immunomodulatory drugs are highly effective agents used in the treatment of multiple myeloma and they have been tested in other monoclonal gammopathies including AL amyloidosis. This review is focused on current experiences with three representatives of immunomodulatory agents: thalidomide, lenalidomide and the most current, pomalidomide (Table 2).

2. Mechanism of action of immunomodulatory drugs Immunomodulatory drugs (IMiDs) are a group of therapeutic compounds that are analogues of thalidomide, a glutamic acid derivate. Thalidomide was first synthesized by Chemie Grünenthal in 5

1954 and was broadly used as an antiemetic agent for morning sickness during pregnancy. In early 1960s its teratogenic effect was recognized and the agent was immedietaly withdrawn from most markets (Rajkumar, 2004). Nevertheless, around the same time, it was serendipitously discovered that thalidomide is effective in the treatment of erythema nodosum leprosum, a disease characterized by high levels of serum TNFα (Iyer et al., 1971). This ability of thalidomide to inhibit production of TNFα by activated human monocytes was presented in 1991 (Sampaio et al., 1991). Besides this antiinflammatory effect, other mechanisms of action were gradually discovered such as its immunomodulatory effect, direct antitumor activity, anti-angiogenic activity and its effect on the bone marrow microenvironment. Its exact mechanism of action, however, has remained unclear for a long time. Recent discoveries have brought to light how these drugs exactly work (Krönke et al., 2014; Lu et al., 2014). All these properties of IMiDs were dominantly investigated in multiple myeloma, the most common malignant monoclonal gammopathy (Quach et al., 2010; Sedlarikova et al., 2012). As AL amyloidosis is also a clonal plasma cell dyscrasia, it is possible to presume that majority of IMiDs´ effects will be identical, although the intensity of each effect may differ in various plasma cell disorders (Bartlett et al., 2004).

2.1. Immunomodulatory effects Survival of malignant plasma cells, as other tumor cells, is partially facilitated by the impaired endogenous immune surveillance against tumor antigens (Zou, 2005). IMiDs are able to affect the immune system in several ways that lead to the eradication of malignant plasma cells. The first effect is co-stimulation of T cells. T cell activation is mediated via antigen specific T cell receptor (TCR), but also requires a co-stimulation signal mostly provided by professional antigen-presenting cells (APC). IMiDs are able to co-stimulate CD3+ T cells that have been partially activated either by anti CD3+ or dendritic cells (DC). The presence of IMiDs abolishes the requirement of the secondary co-stimulation signal from APCs (Davies et al., 2001). IMiDs improve proliferation of both CD4+ and CD8+ T cells and augment the production of Th2 cytokines – interleukin 2 (IL-2) and interferon γ (IFN-γ). The higher production of IL-2 in T cells is due to the depletion of IKZF1 and IKZF3, but this exact mechanism will be discussed later (Krönke et al., 2014). Higher levels of IL-2 and IFN-γ increase the number of natural killer cells (NK cells), improve their function and mediate the death of malignant plasma cells (Sedlarikova et al., 2012). Natural killer T cells (NKT cells) are T lymphocytes which bear natural killer cell markers on their surfaces and they possess a direct tumor cytotoxic effect. NK cells have an irreplacable role in innate immunity, in killing both tumor and virus infected cells. The presence of thalidomide, lenalidomide and pomalidomide enhance both NKT and NK cells that lead to the death of clonal plasmocytes (Davies et al., 2001; Quach et al., 2010).

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Another possible effect of IMiDs is the inhibition of regulatory T cells (Tregs) that probably play an important role in establishing and maintaining immunological unresponsiveness to selfantigens. Several studies have shown that Tregs are expanded in hematological malignancies and also in solid tumors and that IMiDs inhibit proliferation of Tregs via decreased FoxP3 mRNA expression (Muthu Raja et al., 2011; Sedlarikova et al., 2012). However, as there are many contradictory results, a conclusion cannot be drawn at this moment.

2.2. Direct antitumor effects Immunomodulatory drugs also possess a direct anti-proliferative effect via the inhibition of the cyclin-dependent kinase (CDK) pathway, downregulation of anti-apoptotic proteins and activation of FAS-mediated cell deathThe IMiD-induced cell cycle arrest of malignant plasmocytes in G1 phase is mediated via increased levels of the p21WAF-1 protein through epigenetic modifications (Hideshima et al., 2000; Sedlarikova et al., 2012). Clonal plasma cells are protected against apoptosis by anti-apoptotic proteins regulated via NF-κB transcription factor (Wang et al., 1998). It was demonstrated that treatment with IMiDs downregulated the activity of this transcription factor in clonal plasmocytes (Chu et al., 1997). The exact mechanism of the antitumor activity of IMiDs has been recently described by several groups of investigators (Krönke et al., 2014; Lu et al., 2014). It seems that IKZF1 (Ikaros) and IKZF3 (Aiolos), two lymphoid transcription factors playing central roles in the biology of B and T cells, are responsible for the antitumor effect of lenalidomide and all immunomodulatory agents. It was shown that lenalidomide causes selective ubiquitination and degradation of these two transciption factors by the CRBN-CRL4 (cereblon-cullin4A-RING) E3 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors for terminal differentiation of B and T cell lineages. Lenalidomide causes increased binding of IKZF1 and IKZF3 to cereblon (CRBN) and promotes their ubiquitination and degradation. It seems that CRBN is essential for lenalidomide activity in multiple myeloma. So, in aggregate it was shown that lenalidomide acts via a previously unknown mechanism of action– enforced binding of the substrate receptor CRBN to IKZF1 and IKZF3, which results in the degradation and depletion of IKZF1 and IKZF3 (Krönke et al., 2014; Licht et al., 2014; Lu et al., 2014).

2.3. Anti-angiogenic activity All IMiDs possess an anti-angiogenic effect, although it is generally considered that this activity prevails in thalidomide whereas lenalidomide and pomalidomide have far greater immune enhancing effects (Dredge et al., 2002). This property of thalidomide is probably dominantly responsible for the development of malformations in children in the late 1950s and early 1960s. It appears that IMiDs modulate chemotactic factors affecting endothelial cell migration such as TNFα, 7

VEGF and ßFGF secreted by bone marrow stromal cells (Dredge et al., 2002). It seems that PI3K/Aktsignalling pathway plays a key role in anti-angiogenic activity of IMiDs because immunomodulatory drugs reduce Akt phosphorylation, thus they affect the expression of these chemotactical factors and therefore restrain angiogenesis (Dredge et al., 2005; Sedlarikova et al., 2012).

2.4. Anti-inflammatory effects The relative contribution of anti-inflammatory properties of IMiDs towards their anti myeloma activity is uncertain (Quach et al., 2010). Cyclooxygenase 2 (COX-2), which catalyzes the conversion of arachidonic acid to various prostaglandins (PG), is involved in the pathogenesis of several malignancies including plasma cell dyscrasias (Masferrer et al., 2000). For instance, PG-E2 promotes tumor induced angiogenesis, production of IL-6 and, of course, inflammation (Hinson et al., 1996). Thalidomide, lenalidomide and pomalidomide are able to inhibit the expression of COX-2 via the shortening of the half life of COX-2 mRNA in a dose dependent manner (Payvandi et al., 2004). Another mechanism of the anti-inflammatory action of IMiDs is their inhibitory effect on TNFα (an inflammatory cytokine produced by monocytes and macrophages), which is mediated via the degradation of TNFα mRNA (Sampaio et al., 1991). In comparison with thalidomide, inhibition of TNFα was 2000x more potent with lenalidomide and 20000x more potent with pomalidomide (Muller et al., 1999). Therefore, immunomodulatory agents may have a therapeutical potential not only in multiple myeloma or other malignancies, but also in inflammatory conditions such as Crohn’s disease or rheumatoid arthritis (Akobeng and Stokkers, 2009; Quach et al., 2010).

2.5. Effects on the bone marrow microenvironment Osteolytic lesions and back pain are usually the first symptoms that force patients with multiple myeloma to visit their physicians. Immunomodulatory drugs possess many properties influencing the bone marrow microenvironment and interactions between malignant plasma cells, including the inhibition of osteoclastogenesis (Breitkreutz et al., 2008) or changes in the expression of adhesive molecules important for the proliferation and migration of plasmocytes (Geitz et al., 1996). In AL amyloidosis, however, osteolytic lesions are extremely rare, so the the detailed description of these mechanisms will not be mentioned in this part.

3. Thalidomide in the treatment of AL amyloidosis Thalidomide is the first and principal drug of the immunomodulatory agents group. In biochemical nomenclature it is called 2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (C13H10N2O4), a synthetic derivate of glutamic acid. Thalidomide exists as a racemic mixture of two optically active

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enantiomers, S and R, wherein the S enantiomer is responsible for the teratogenic and anti-tumor effect, while the R enantiomer possesses a sedative effect (Rajkumar, 2004). Low dose thalidomide used as monotherapy in the treatment of AL amyloidosis is not very efficient (Dispenzieri et al., 2003; Seldin et al., 2003). On the contrary, high doses of thalidomide are not well tolerated in this fragile group of patients (Dispenzieri et al., 2003) either. The combination of thalidomide and dexamethasone achieved 48% of hematological responses and 19% of hematological complete remissions in the group of patients with relapsed/refractory AL (Palladini et al., 2005). The use of thalidomide and dexamethasone as adjuvant chemotherapy after ASCT in patients with newly diagnosed AL, who had not obtained CR after ASCT, was also investigated. The benefits of adjuvant therapy were seen in 42% (13/31) of patients who had an improvement in hematological response at 12 months post-SCT, including seven who achieved a CR after having stable disease (SD) or partial remission (PR) initially (Cohen et al., 2007). Patients with advanced cardiac AL amyloidosis (NYHA IV), who received a combination of oral melphalan, thalidomide and dexamethasone, reached hematological response in 36%, but 27% of them died during treatment (Palladini et al., 2009). The combination of another alkylator, cyclophosphamide, with thalidomide and dexamethasone (regimen CTD) was used in 75 transplant ineligible patients with advanced AL amyloidosis, including 44 patients (56.6%) with clonal relapse to prior therapy. Hematological response occurred in 48 out of 65 (74%) evaluable patients, including a complete response in 14 (21%) patients. The median estimated OS from commencement of treatment was 41 months (Wechalekar et al., 2007). The ALChemy trial, one of the largest prospective observational studies in AL amyloidosis worldwide, is currently being conducted at the UK Amyloidosis Centre in London. Recently, the update of this study was presented in the XIVth International Symposium on Amyloidosis. The data of 500 enrolled patients showed that 58% of patients were treated with a thalidomide-based regimen (mainly CTD), 51% of all patients reached a hematological response with a complete response in 14%. The estimated survival at 12, 36 and 48 months was 67%, 56% and 51%, respectively. Toxicity grade 3 or higher was seen in 59% of all patients (Gillmore et al., 2012; Wechalekar et al., 2014). These results show that the toxicity of this regimen remains high and in the UK bortezomib–based regimens are being used increasingly. A retrospective matched comparison of cyclophosphamide, bortezomib and dexamethasone (CVD) versus risk-adapted cyclophosphamide, thalidomide and dexamethasone was published recently by the same London group. The overall response rates were 71.0% vs 79.7% in the CVD and CTD arms, respectively, (p = 0.32). A higher CR rate was observed in the CVD arm (40.5%) vs. CTD (24.6%), (p = 0.046). 1 year OS was 65.2% and 66.7% for CVD and CTD, (p= 0.87) and the median PFS was 28.0 and 14.0 months for CVD and CTD, respectively (p = 0.039). In summary, CVD correlated with improved depth of response and superior PFS thus supporting its use in the frontline setting, but there was no difference in the terms of OS (Venner et al., 2014). The toxicity of thalidomide containing regimens is not negligible. Toxicities of grade 3 and higher were present in more than 50% of treated patients practically in all published studies. Fluid 9

retention, symptomatic bradycardia, peripheral neuropathy and progression of renal failure are the most common (Gillmore et al., 2012; Palladini et al., 2005; Seldin et al., 2003). Thalidomide in combination with other drugs shows clinical effectivity but its toxicity is limiting. These combinations represent the treatment modality for patients refractory to bortezomib or for patients who tolerate thalidomide’s toxic effects.

4. Lenalidomide in the treatment of AL amyloidosis Lenalidomide,3-(4-amino-l-oxo-l,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione (C13H13N3O3), has a preserved chiral centre and occurs as a racemate, in a mixture of two optically active enantiomers, S and R, similar to thalidomide (Kotla et al., 2009). Lenalidomide (Revlimid, formerly

CC-5013)

is

ranked

amongst

thalidomide’s

analogues

(Celgene’s

class

of

immunomodulatory compounds) with a more favorable toxic profile and increased efficacy. This second generation IMiD showed approximately 50 – 2000x higher effectivity depending on the type of “in vitro“ test compared to thalidomide. Unfortunately, its teratogenic potential is preserved (Quach et al., 2010; Sedlarikova et al., 2012). Lenalidomide in monotherapy is not an appropriate treatment modality for patients with AL amyloidosis because of low efficacy and inadequate toxicity, especially in patients with renal insufficiency. Initial doses of lenalidomide should not exceed 15 mg/day (Adam et al., 2013; Dispenzieri et al., 2007). In 2007, two phase-II studies with a combination of lenalidomide and dexamethasone (Len/Dex) were conducted mostly in relapsed/refractory patients with AL. The hematological overall response rates ranged from 41% to 67% (Dispenzieri et al., 2007; Sanchorawala et al., 2007b). The most common treatment-related adverse event (AE) was myelosupression in 45% and 35% of patients, respectively. Fatigue, skin rash and thromboembolic complications represent other frequent side effects. Another study with Len/Dex was published in 2012 by Palladini et al., in which this combination was used as salvage therapy for 24 patients refractory to bortezomib, melphalan and in 79% also to thalidomide. Hematological response was observed in 41% of patients, but 50% of patients experienced severe adverse events (Palladini et al., 2012b). The most recent study with Len/Dex was conducted in 84 AL amyloidosis patients with relapsed/refractory clonal disease following prior treatment with thalidomide (76%) and/or bortezomib (68%). The overall hematological response rate was 61%, including 20% of complete responses. While the median OS has not been reached, the 2 year OS and PFS rates were 84% and 73%, respectively. 16% of patients achieved an organ response at 6 months, with a marked improvement in organ responses in patients on long term therapy (median duration 11 months) and 55% achieving renal responses by 18 months. The rate of renal responses among patients who received prolonged treatment was unexpectedly high, raising the possibility that immunomodulatory effects of lenalidomide therapy might enhance the otherwise slow natural regression of amyloid deposits (Mahmood et al., 2014b). The combination of 10

Len/Dex showed clinical activity in the pre-treated group of patients with AL amyloidosis and represent a valuable option as salvage therapy, but the treatment is accompanied by significant toxicity (Gatt and Palladini, 2013; Palladini et al., 2012b). The complete response rate remained low (0 – 29%) with the Len/Dex regimen, therefore a number of phase II studies have focused on the addition of an alkylator to this combination. Three independent clinical trials with incorporated cyclophospamide were conducted (regimen CLD) (Kastritis et al., 2012; S. K. Kumar et al., 2012; Palladini et al., 2013). The hematological response rates ranged from 55% to 62% and CR rates remained low - approximately 10%. The toxicity was prominent in all these studies with more than 60% of patients reporting severe adverse events, including mainly hematological toxicity in approximately 40% of cases. Fluid retention, fatigue, skin rash and gastrointestinal problems rank amongst other frequent side effects. Results from another clinical trial with CLD regimen in 28 newly diagnosed AL amyloidosis patients not eligible for ASCT were recently presented by Cibeira et al. The treatment schedule consists of 12 cycles of CLD followed by maintentance with low-dose lenalidomide (10mg) and dexamethasone until intolerance or disease progression. The overall hematological response rate was 46%, including complete response in 14%. The organ response rate was 32%. Serious adverse events were reported in 25% of cases (Cibeira et al., 2014). The addition of melphalan to lenalidomide and dexamethasone (regimen MLD) is other logical combination considering that the regimen Mel/Dex has been the golden standard of the treatment of AL amyloidosis to date. In the study published by Moreau et al., 26 patients with newly diagnosed AL were treated with the MLD regimen, hematological response was observed in 58% of patients with CR in 23%. The maximum tolerated dose of lenalidomide was 15 mg/day and the dose of melphalan was 0.18 mg/kg/day (Moreau et al., 2010). In another study with MLD in a more pretreated group of patients, hematological partial and complete responses were achieved by 43% and 7% of patients, respectively. Grade 3/4 toxicities were experienced by 88% patients, with myelosuppression being the most common (Sanchorawala et al., 2013). The largest phase II clinical study using lenalidomide in AL amyloidosis was presented at ASH 2013 by Schönland and his colleagues from Heidelberg (Bochtler et al., 2013). 50 patients with newly diagnosed AL amyloidosis ineligible for HDM-ASCT were enrolled in this prospective single center trial. The treatment schedule was 6 cycles of oral treatment with lenalidomide 10 mg/day, days 1-21, melphalan 0.15 mg/kg/day, days 1-4 and dexamethasone 20 mg/day, days 1-4, every four weeks. Hematological response was achieved in 78% of patients: CR in 9 (20%) and PR in 26 (58%) of 45 evaluable patients, respectively. Organ response was observed in 5 (10%) patients at the end of the study (6 months after the end of treatment). In comparison to the historical Mel/Dex group from the same center (patients with the same inclusion and exclusion criteria), there was a significantly higher hematological response rate in the MLD group (78% vs. 58%, p=0.06) and the improvement was also seen in median OS (not reached vs. 26 months, p=0.03). Overall, toxicity was manageable in most 11

patients, the most common being hematological toxicity (neutropenia in 76% patients). The most common non-hematological AE was the worsening of cardiac function or symptoms of autonomic neuropathy (14 patients) and infection (8 patients). Treatment with MLD was effective and feasible in this cohort of mostly elderly patients including patients with advanced cardiac involvement (Bochtler et al., 2013). The use of lenalidomide in combination with an alkylator in these pre-treated groups of patients with AL amyloidosis was frequently associated with toxicities grade 3/4, dominantly with hematological toxicity and fatigue (Bochtler et al., 2013; Kastritis et al., 2012; S. K. Kumar et al., 2012; Palladini et al., 2013; Sanchorawala et al., 2013). The higher frequency of severe adverse events is expectable considering the fragility of this population of patients and the fact that these chemotherapeutical regimens have not been optimized yet. The results of the recent clinical trial conducted by Schönland et al. show that lenalidomide possesses a high therapeutic potential in patients with AL amyloidosis. Accompanied toxicity may be associated with a different metabolic pathway of lenalidomide in comparison with thalidomide and pomalidomide. The pharmacokinetics of lenalidomide is connected with renal function, because the excretion of the drug is provided dominantly by the kidneys. The regimens containing lenalidomide may be a good treatment alternative for patients refractory to bortezomib or for patients with polyneuropathy. The use of lenalidomide in the first line treatment in AL amyloidosis should be investigated and its use should be optimized in clinical trials, especially in patients with no renal insufficiency.

5. Pomalidomide in the treatment of AL amyloidosis Pomalidomide (CC-4047, Actimid, Imnovid, Pomalyst) is the third immunomodulatory agent used in clinical practice. The chemical formula of pomalidomide is 4-amino-2-(2,6-dioxo-3piperidyl)isoindoline-1,3-dione (C13H11N3O4). This compound is derived from thalidomide by adding an amino group to position 4 of the phthaloyl ring and exists in two forms, enantiomers S and R (Sedlarikova et al., 2012). This thalidomide analogue possesses pleiotropic effects on myeloma cells. Most of these mechanisms are probably effective also on clonal plasmocytes in AL amyloidosis (Quach et al., 2010). Pomalidomide inhibits angiogenesis, induces apoptosis of multiple myeloma cells, has strong immunomodulatory abilities and is the most effective in TNF-mRNA degradation in comparison to thalidomide and lenalidomide (Dredge et al., 2002; Muller et al., 1999). Pomalidomide is the newest immunomodulatory agent used in the treatment of AL amyloidosis. Pomalidomide and dexamethasone (regimen Pom/Dex) were investigated in the treatment of this disease, but only in a limited number of patients. A phase II clinical trial with 33 patients with refractory/relapsed AL amyloidosis was conducted by investigators from Mayo Clinic Rochester (Dispenzieri et al., 2012). Patients were treated with pomalidomide 2 mg/day for 28 days (1 cycle) along with orally administered dexamethasone 40 mg/week. A majority (82%) of patients had 12

cardiac involvement, 25% had Mayo cardiac stage III disease. The hematological response was rapid (median 1.9 months) and was seen in 48% of patients with 3% of complete response, organ treatment response was achieved in 18% of patients. Remarkably, hematological responses to pomalidomide were seen in 40 - 50% of patients pre-treated with lenalidomide or bortezomib. The median overall survival rate was 27.9 months with a 1year OS rate of 76%. Median and 3year PFS were 14.1 months and 17%, respectively. The toxicity profile of the regimen was manageable, although grade 3 or higher AEs at least possibly related to treatment were reported in 52% (non-hematological) and 91% (hematological) (Dispenzieri et al., 2014, 2012). The NT-proBNP reduction did not follow the hematological response similar to what has been seen in other studies of patients with AL amyloidosis taking immunomodulatory drugs (Dispenzieri et al., 2010; Gibbs et al., 2009; Tapan et al., 2010). A second clinical trial with high dose (4 mg/day) pomalidomide and oral dexamethasone (40 mg/week) was conducted by Milani et al. and its preliminary results were presented at ASH 2013. 27 patients with relapsed/refractory AL previously exposed to an alkylator or bortezomib were enrolled in the trial and 41% had also received an immunomodulatory agent. Overall, 18 patients (67%) achieved a hematological response, with 5 (18%) very good partial responses. Moreover, of the 7 patients previously exposed to lenalidomide, 5 responded to pomalidomide and the 3 patients who were refractory to ixazomib responded as well, indicating that pomalidomide can overcome resistance to other immunomodulatory drugs and a second-generation proteasome inhibitor. Overall, 18 patients (67%) experienced severe (grade 3-4) adverse events: neutropenia (n=8, 30%), fluid retention (n=7, 26%, all with heart involvement), infection (n=3, 11%), less common were neuropathy and rash. As previously reported with other immunomodulatory drugs, pomalidomide was associated with an increase in NT-proBNP during cycle 1 (Milani et al., 2013). The preliminary data of the most recent clinical trial with Pom/Dex in 12 patients with relapsed AL were presented at the XIVth International Symposium on Amyloidosis by Sanchorawala et al. Hematological complete response was observed in 2 of 9 evaluable patients (22%), toxicity grade 3 or higher was reported in 6 cases (50%). Other data were not specified but the trial is still ongoing (Sanchorawala et al., 2014b). These results show good efficacy of pomalidomide and dexamethasone in patients with refractory AL amyloidosis previously exposed also to other IMiDs. Further studies to optimize this potent rescue regimen are warranted.

6. Problematics of response assessment with IMiDs NT-proBNP and BNP are important and useful biomarkers of cardiac involvement, especially of heart failure. The prognostic value of these biomarkers in AL amyloidosis is indisputable, but they also serve to assess the organ (cardiac) treatment response. Nevertheless, the use of immunomodulatory agents may cause an elevation of cardiac biomarkers in a significant proportion of 13

patients usually during the first cycle of chemotherapy. This phenomenon may complicate the assessment of organ treatment response in patients treated with IMiDs. Gibbs et al. described this phenomenon in 51 patients who achieved complete hematological response measured by the decrease of serum free light chains (FLC). Overall, 42 patients were treated with a regimen based on thalidomide (CTD) and 9 patients were treated with melphalan and dexamethasone (Mel/Dex). An increase in NT-proBNP from baseline was seen in 36 (71%) patients at 6 months. A subsequent fall in their NT-proBNP levels was seen at 12 months in 33 (92%) of these patients. There was no significant rise in creatinine serum level at 6 months, suggesting that the rise in NT-proBNP was not due to a change in the renal function. Importantly, there was no difference in the rate of NT-proBNP rise when comparing the CTD regimen to Mel/Dex. It is clearly shown that NTproBNP values can rise transiently in the immediate post chemotherapy period in nearly three quarters of patients with a complete FLC response in this analysis. The exact mechanism of this phenomenon has remained unclear and it has not impacted the rate of clonal relapse or overall survival (Gibbs et al., 2009). Tapan et al. described the rise of BNP level (> 30% increase from baseline) in 86% of patients treated with lenalidomide and dexamethasone (Len/Dex). In 68% of patients the increase occured during the first cycle of chemotherapy. All the patients with an increase in BNP were asymptomatic without association of modification in NYHA class congestive heart failure (Tapan et al., 2010). Similarly, the results of clinical trials with pomalidomide showed the discrepancy between an increase of cardiac biomarkers and hematological response (Dispenzieri et al., 2012; Palladini et al., 2013). Even though this phenomenon is transient, it complicates the assessment and interpretation of treatment organ response in AL. It is useful to evaluate the dynamics of FLC levels and levels of cardiac biomarkers. Patients with AL amyloidosis receiving IMiDs whose cardiac biomarkers rise should not be assumed to be failing therapy without other signs of disease progression, but should be monitored closely. Clinical hematologist-oncologists must carefully consider how to evaluate the organ treatment response, dominantly when the decision of changing therapy is about to be made. Further data is needed for non-thalidomide based regimens to assess if this has an effect to the same extent.

7. Treatment strategies in AL amyloidosis and role of IMiDs There is no consensus of optimal therapy for patients with newly diagnosed AL amyloidosis, because there is minimum data available from randomized clinical trials, because of the rarity of this disease and the small number of patients possibly enrolled. Often, the treatment strategy is based on expert opinions at a national, European or global level. None of the 3 published randomized clinical trials include novel agents such as proteasome inhibitors or immunomodulatory agents. Thus, the most evident benefit of novel agents is increased flexibility of our treatment strategy in newly diagnosed patients, because we can switch from a thalidomide-based to bortezomib-based regimen if 14

not tolerated and/or effective. Moreover, we have now got several effective treatment options in a relapse setting. The treatment strategy in patients with AL amyloidosis is shaped by background factors including performance status, age, comorbidities, contraindications to drugs and also patient preference. Risk stratification adopted from the Mayo Clinic staging system is crucial in the choice of treatment strategy and may be a helpful instrument for physicians as many of the patients are very fragile. According to this risk adapted staging system based on the level of cardiac biomarkers (NTproBNP and Troponin T), patients with AL are divided into three groups (Dispenzieri et al., 2004; S. Kumar et al., 2012). ASCT should always be considered as a part of front-line therapy in low-risk patients with AL amyloidosis. These patients can be also treated with a combined bortezomib based regimen which allows the safe harvest of peripheral stem cells and leaves the performance of ASCT only in the case of not achieving complete remission (Mikhael et al., 2012; Venner et al., 2012). Approximately 50% of patients with newly diagnosed AL are in the intermediate risk group, these patients are not suitable for undertaking ASCT as front line treatment. According to two recently published retrospective case control studies, there is still no certainty about the best treatment option for this group of patients. Bortezomib-based regimens yielded higher complete response rates, and these responses were rapidly achieved, but there was no clear improvement in overall survival. It was only in an Italian study where one subgroup of patients with New York Heart Association class of < 3 and NT-proBNP of ≤ 8500 ng/l, who were not fit enough to recieve high dose dexamethasone, had a superior OS when bortezomib was added to Mel/Dex (G. Palladini et al., 2014; Venner et al., 2014). The results of the randomized phase III trial are still awaited. The last interim analysis from ASH 2014 indicates that the addition of bortezomib to Mel/Dex grants more profound hematologic responses that should be balanced with a relative increase in toxicity (Merlini et al., 2014). Nevertheless, also thalidomide based regimens may be successfuly used in a selected cohort of patients. A high-risk group of patients has a poor prognosis dominantly because of extensive organ involvement and their treatment remains problematic with the use of low-dose corticosteroids in combination with novel agents (Mahmood et al., 2014a). According to recent data published by Wechalekar et al. at EHA 2014, it seems that thalidomide based regimens (TMD) are slightly beneficial compared to bortezomib based regimens with an estimated survival rate of 75% vs. 61% at four months for patients with a high-risk Mayo Clinic stage (Wechalekar et al., 2014). Immunomodulatory agents showed good efficacy in the treatment of AL and they may be used mainly in patients with relapsed/refractory disease but also as a part of first-line therapy. The optimization of the regimen reflecting its toxicity plays an important role. Due to the lack of randomized clinical trials, it is difficult to decide which immunomodulatory drug is better. Pomalidomide may be a representative agent of this group with a good balance between high effectivity and tolerable toxicity. The summary of practically all published clinical trials containing immunomodulatory agents is in Table 3. 15

Thalidomide in combination with other drugs showed good clinical activity in the treatment of AL, but its use is accompanied by significant toxicity. This IMiD may be a treatment alternative for patients refractory to bortezomib or may be used also as a first line treatment if well tolerated. Lenalidomide may be used also in patients refractory to bortezomib or in patients with neuropathy. The toxicity of lenalidomide-containing regimens may be a problem, especially for patients with renal insufficiency. Pomalidomide proved to have high efficacy in pilot studies and the lowest toxicity of all immonomodulatory agents and seems to be a very promising drug in the treatment of AL. The appropriate dosage of IMiDs is important and it is possible to recommend 50–100 mg/day of thalidomide, 10–15 mg/day of lenalidomide, 2 mg/day or 4 mg/day of pomalidomide (based on available results of clinical studies). Concerning pomalidomide, it is not possible to decide which dose level is better, because only two clinical trials have been conducted to date. Combinations of immunomodulatory drugs and a corticosteroid well tolerated in multiple myeloma may represent an appropriate treatment option also in AL amyloidosis.

8. Conclusion Immunoglobulin light chain amyloidosis is a rare disease, but the most common of the systemic amyloidoses. Patients are often diagnosed in an advanced stage with multiorgan involvement and their prognosis remains poor. Nevertheless, recently published data suggests that overall survival of patients eligible for ASCT with preserved cardiac function is surprisingly long and with a life expectancy much longer than in multiple myeloma patients. The combination of melphalan and dexamethasone is still a very good treatment option for transplant ineligible patients, but novel agents may offer some benefits. Proteasome inhibitors and mainly combined bortezomib based regimens seem to be a good choice in front-line therapy. However, thalidomide based regimens may be used in this setting as well, if well tolerated. Any data of randomized clinical trials, which would support the use of one or another group of agents are still not available. Recent trials with immunomodulatory agents, on which this review is focused, show a good clinical efficacy, but the toxicity remains significant in this fragile group of patients. Pomalidomide seems to be a very promising drug with preserved high effectivity and favorable toxic profile in patients with renal insufficiency as well. The development of a new generation of proteasome inhibitors, e.g. oral ixazomib (MLN9708), gives physicians more options in the decision making process of adequate treatment approach. As AL amyloidosis is a very uncommon disorder, most patients should be treated within the clinical trials. Active clinical trials with immunomodulatory drugs in AL amyloidosis are summarized in Table 4. Also, novel therapies targeted directly on amyloid and not on pre-existing malignant clone of plasmocytes may play a role in the future. Recently, the clinical trial phase III with NEOD001 has been initialized. NEOD001 is a monoclonal antibody that targets the amyloid that affects both AL and 16

AA amyloidosis. Finally, what seems to be the most important issue in clinical practise is an increasing awareness of this rare disease amongst physicians - dominantly cardiologists, nephrologists and also hematologists.

Conflict of interest statement The authors have no conflict of interest to be disclosed.

Acknowledgements TJ – wrote the paper ZK – proofread the paper RH – concept, overall proofread, work coordination

This work was supported by the Moravian-Silesian Region grants MSK 02680/2014/RRC and MSK 02692/2014/RRC; grants by MH CZ-DRO-FNOs/2014; by The Ministry of Education, Youth and Sports (Institutional Development Plan of University of Ostrava in 2015); projects SGS01/LF/2014-2015, SGS02/LF/2014-2015, SGS03/LF/2015-2016 and by grant IGA of The Ministry of Health 15-29667A. The authors want to give special thanks to Eva Jarosova for administrative support.

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bortezomib in patients with relapsed systemic AL amyloidosis: results of a phase 1/2 study. Blood 118, 865–873. doi:10.1182/blood-2011-02-334227 Sampaio, E.P., Sarno, E.N., Galilly, R., Cohn, Z.A., Kaplan, G., 1991. Thalidomide selectively inhibits tumor necrosis factor alpha production by stimulated human monocytes. J. Exp. Med. 173, 699–703. Sanchorawala, V., Doros, G., Quillen, K., Sloan, J.M., Shelton, A.C., Brauneis, D., Finn, K.T., Seldin, D.C., 2014a. Long-Term Outcome of Patients with AL Amyloidosis Treated with High-Dose Melphalan and Stem Cell Transplantation: 19 Year Experience at a Single Center. Biol. Blood Marrow Transpl. 20, S47–S48. doi:10.1016/j.bbmt.2013.12.045 Sanchorawala, V., Patel, J.M., Sloan, J.M., Shelton, A.C., Zeldis, J.B., Seldin, D.C., 2013. Melphalan, lenalidomide and dexamethasone for the treatment of immunoglobulin light chain amyloidosis: results of a phase II trial. Haematologica 98, 789–792. doi:10.3324/haematol.2012.075192 Sanchorawala, V., Shelton, A., Stephen, L., Sloan, J.M., Seldin, D.C., 2014b. Pomalidomide and dexamethasone in patients with relapsed light-chain amyloidosis (AL): Results of a phase 1 study. Presented at the XIVth International Symposium on Amyloidosis, Indianapolis. Sanchorawala, V., Skinner, M., Quillen, K., Finn, K.T., Doros, G., Seldin, D.C., 2007a. Long-term outcome of patients with AL amyloidosis treated with high-dose melphalan and stem-cell transplantation. Blood 110, 3561–3563. doi:10.1182/blood-2007-07-099481 Sanchorawala, V., Wright, D.G., Rosenzweig, M., Finn, K.T., Fennessey, S., Zeldis, J.B., Skinner, M., Seldin, D.C., 2007b. Lenalidomide and dexamethasone in the treatment of AL amyloidosis: results of a phase 2 trial. Blood 109, 492–496. doi:10.1182/blood-2006-07-030544 Sedlarikova, L., Kubiczkova, L., Sevcikova, S., Hajek, R., 2012. Mechanism of immunomodulatory drugs in multiple myeloma. Leuk. Res. 36, 1218–1224. doi:10.1016/j.leukres.2012.05.010 Seldin, D.C., Choufani, E.B., Dember, L.M., Wiesman, J.F., Berk, J.L., Falk, R.H., O’Hara, C., Fennessey, S., Finn, K.T., Wright, D.G., Skinner, M., Sanchorawala, V., 2003. Tolerability and efficacy of thalidomide for the treatment of patients with light chain-associated (AL) amyloidosis. Clin. Lymphoma 3, 241–246. Skinner, M., Anderson, J., Simms, R., Falk, R., Wang, M., Libbey, C., Jones, L.A., Cohen, A.S., 1996. Treatment of 100 patients with primary amyloidosis: a randomized trial of melphalan, prednisone, and colchicine versus colchicine only. Am. J. Med. 100, 290–298. Skinner, M., Sanchorawala, V., Seldin, D.C., Dember, L.M., Falk, R.H., Berk, J.L., Anderson, J.J., O’Hara, C., Finn, K.T., Libbey, C.A., Wiesman, J., Quillen, K., Swan, N., Wright, D.G., 2004. High-dose melphalan and autologous stem-cell transplantation in patients with AL amyloidosis: an 8-year study. Ann. Intern. Med. 140, 85–93. Tapan, U., Seldin, D.C., Finn, K.T., Fennessey, S., Shelton, A., Zeldis, J.B., Sanchorawala, V., 2010. Increases in B-type natriuretic peptide (BNP) during treatment with lenalidomide in AL amyloidosis. Blood 116, 5071–5072. doi:10.1182/blood-2010-09-305136 Venner, C.P., Gillmore, J.D., Sachchithanantham, S., Mahmood, S., Lane, T., Foard, D., Rannigan, L., Gibbs, S.D.J., Pinney, J.H., Whelan, C.J., Lachmann, H.J., Hawkins, P.N., Wechalekar, A.D., 2014. A matched comparison of cyclophosphamide, bortezomib and dexamethasone (CVD) versus risk-adapted cyclophosphamide, thalidomide and dexamethasone (CTD) in AL amyloidosis. Leukemia 28, 2304–2310. doi:10.1038/leu.2014.218 Venner, C.P., Lane, T., Foard, D., Rannigan, L., Gibbs, S.D.J., Pinney, J.H., Whelan, C.J., Lachmann, H.J., Gillmore, J.D., Hawkins, P.N., Wechalekar, A.D., 2012. Cyclophosphamide, bortezomib, and dexamethasone therapy in AL amyloidosis is associated with high clonal response rates and prolonged progression-free survival. Blood 119, 4387–4390. doi:10.1182/blood-2011-10-388462 Wang, C.Y., Mayo, M.W., Korneluk, R.G., Goeddel, D.V., Baldwin, A.S., 1998. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281, 1680–1683. Wechalekar, A.D., Goodman, H.J.B., Lachmann, H.J., Offer, M., Hawkins, P.N., Gillmore, J.D., 2007. Safety and efficacy of risk-adapted cyclophosphamide, thalidomide, and dexamethasone in systemic AL amyloidosis. Blood 109, 457–464. doi:10.1182/blood-2006-07-035352 Wechalekar, A.D., Schonland, S.O., Kastritis, E., Gillmore, J.D., Dimopoulos, M.A., Lane, T., Foli, A., Foard, D., Milani, P., Rannigan, L., Hegenbart, U., Hawkins, P.N., Merlini, G., Palladini, G.,

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2013. A European collaborative study of treatment outcomes in 346 patients with cardiac stage III AL amyloidosis. Blood 121, 3420–3427. doi:10.1182/blood-2012-12-473066 Wechalekar, A., Foard, D., Rannigan, L., Lane, T., Sachachtanatham, S., Mahmood, S., Sayed, R., Patel, K., Whelan, C., Lachmann, H., Hawkins, P., Gillmore, J., 2014. Characteristics and outcomes of 714 patients with systemic AL amyloidosis – analysis of a prospective study (ALChemy study). Presented at the XIVth International Symposium on Amyloidosis, Indianapolis. Zou, W., 2005. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer 5, 263–274. doi:10.1038/nrc1586

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Biography Tomas Jelinek is a physician at the Department of Haematooncology at the University Hospital Ostrava, Czech Republic. In 2011 he earned his degree at Masaryk University in Brno, Czech Republic. During his studies at university he conducted internships at Hospital Anna Costa, Santos, Brazil; Universidade de Coimbra, Portugal and Autonomous University of Zacatecas, Mexico. He started his career at the Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and since 2013 he has been working at the Intensive Care and Transplant Unit at University Hospital Ostrava. Dr. Jelinek is a member of Czech Society of Hematology and Czech Myeloma Group. He has several publications in the field of hematological malignancies. His current research interest includes multiple myeloma and other plasma cell disorders. He is also interested in modern diagnostic methods, dominantly flow cytometry and its clinical and scientific application.

Roman Hajek is Professor of Oncology and Vice-rector at the Ostrava University and Head of the Department of Haematooncology at the University Hospital Ostrava, Czech Republic. After qualifying in medicine in 1988 from J. E. Purkyne (Masaryk) University, Faculty of Medicine Brno, Dr. Hajek received a PhD in hematology in 1995 and completed degrees in Internal Medicine in 1996 and in Medical Oncology in 1998. Dr. Hajek was a founder member of the Czech Myeloma Group. He is an active chairman of the Czech Myeloma Group, main investigator of the randomized clinical trials phase III of Czech Myeloma Group and phase I/II/III of other clinical trials in MM including experimental immunotherapy and main investigator of more than 30 national grants. He is a member of several scientific societies, including the European Hematology Association and the Amercian Society of Hematology. He has published over 300 scientific articles and book chapters and he is an Editor of the European Journal of Hematology. His research interests include multiple myeloma, bone marrow transplantation and amyloidosis.

25

Table 1. Hematological and organ response and progression criteria

Criteria CR VGPR PR NR NT-proBNP cTn NYHA class response

Response

Progression

Hematological assessment Normal serum free light chain ratio with negative Any detectable monoclonal protein or abnormal serum and urinary immunofixation FLC ratio (FLC must double) The difference in the free light chains (dFLC) less than 40 mg/l A reduction in the dFLC greater than 50% 50% increase in serum M-protein to > 0.5 g/dl or 50% increase of 24h urine protein to > 200 mg/day A less than 50% response in dFLC FLC increase of 50% to > 10 mg/dl Heart > 30% and > 300 ng/l decrease if baseline > 30% and > 300 ng/l increase NT-proBNP ≥ 650 ng/l ≥ 33% increase ≥ two-class decrease if baseline NYHA class 3 or 4 EF (≥ 10% decrease)

Other organs 50% decrease (at least 0.5 g/day) of 24h urine 50% increase (at least 1 g/day) of 24h urine protein protein (pre-treatment urine protein to > 1 g/day must be > 0.5 g/day) OR Creatinine and creatinine clearance must not 25% worsening of serum creatinine or creatinine worsen by 25% over baseline clearance 50% decrease in abnormal ALP value 50% increase of ALP above the lowest value Liver Decrease in liver size radiographically at least 2 cm Improvement in electromyogram nerve Progressive neuropathy by electromyography or Peripheral nerve conduction velocity nervous system conduction velocity (rare) ALP - alkaline phosphatase; cTn - cardiac troponin; dFLC - difference between involved and uninvolved free light chain; EF - ejection fraction; NR - no response; VGPR - very good partial response Kidney

26

Table 2. Basic characteristics of IMiDs Drug

Structure

Chemical formula

Frequent AEs

Prophylaxis of VTE

Trade name

EMA/FDA approval*

+++

Peripheral polyneuropathy, VTE, obstipation, drowsiness

YES

Inmunoprin, Talidex, Talizer, Thalomid, Myrin

NO/NO

YES

Revlimid

NO/NO

YES

Pomalyst, Imnovid

NO/NO

Teratogenicity Neuropathy

+++

Thalidomide

C13H10N2O4

Lenalidomide

C13H13N3O3

+++

+

Neutropenia, thrombocytopenia, VTE, fatigue, skin rash

Pomalidomide

C13H11N3O4

+++

+

Neutropenia, anemia, thrombocytopenia, VTE

*in AL amyloidosis indication VTE – venous thromboembolism

27

Table 3. Results of clinical trials including IMiDs in AL amyloidosi

Number of patients Regimen (% - newly diagnosed)

Hematologic response (%) (CR [%])

Organ response (%)

The most common adverse events grade 3/4 (%)

Median OS (months) or 1-3 year survival (%)

Thalidomide containing regimens Thalidomide

16

25%

Seldin et al, 2003

(0%)

(0%)

Thal/Dex

31

48%

Palladini et al, 2005

(0%)

(19%)

CTD

75

74%

Wechalekar et al, 2007

(41%)

(21%)

MTD

22

36%

Palladini et al, 2009

(73%)

(5%)

CTD

69

80

Venner et al, 2014

(100%)

(25%)

0%

Overall 50%

Not specified

Fatigue/sedation 38% 26%

Overall 65%

Not specified

Symptomatic bradycardia (26%) 33%

Overall 32%

41

18%

Overall 27%

5.3

27%

Not specified

67% (1 year OS)

Overall 86%

Not specified

Lenalidomide containing regimens Len/Dex

22

41%

23%

28

Dispenzieri et al, 2007

(41%)

(not specified)

Len/Dex

34

67%

Sanchorawala et al, 2007b

(9%)

(29%)

Len/Dex

24

41%

Palladini et al, 2012b

(0%)

(0%)

Len/Dex

84

61%

Mahmood et al, 2014

(0%)

(20%)

CLD

35

60%

Kumar et al, 2012b

(69%)

(11%)

CLD

21

62%

Palladini et al, 2013

(0%)

(5%)

CLD

37

55%

Kastritis et al, 2012

(65%)

(8%)

CLD

28

46%

Cibeira et al, 2015

(100%)

(14%)

MLD

26

58%

Neutropenia 45% 41%

Myelosuppression 35%

Not specified

Fatigue 35%

4%

Overall 50%

14

Thrombocytopenia 17% Not specified

Not specified

84% (2 year OS)

31%

Overall 74%

37.8

Neutropenia 40% 19%

Overall 57%

36

22%

Neutropenia 34%

41% (2 year OS)

32%

Overall 25%

Not specified (study ongoing)

50%

Overall 81%

81% (2 year OS)

29

Moreau et al, 2010

(100%)

(23%)

Neutropenia 11%

MLD

50

78%

10%

Bochtler et al, 2013

(100%)

(20%)

(at six months)

Neutropenia 76%

MLD

16

50%

11%

Overall 88%

Sanchorawala et al, 2013

(69%)

(7%)

Not achieved

Not achieved

Myelosuppression 57%

Pomalidomide containing regimens Pom/Dex

33

48%

Dispenzieri et al, 2012

(0%)

(3%)

Pom/Dex

27

67%

(0%)

(18% - VGPR)

Pom/Dex

12

Not specified

Sanchorawala et al, 2014b

(0%)

(22%)

Milani et al, 2013

15%

Hematologic 91%

27.9

Non-hematologic 52% (short-term follow-up) Not specified

Overall 67% Neutropenia 30% Overall 50%

(short-term follow-up) Not specified (study ongoing)

30

Table 4. Ongoing clinical trials with IMiDs in AL amyloidosis

Regimens Title Experimental arm Pomalidomide orally 1mg/day, days 1-21; A Phase I/II Clinical Trial of Pomalidomide With Melphalan orally 9mg/m2/day days 1-4; Melphalan and Dexamethasone in Patients With Dexamethasone orally 40mg/day days 1Newly Diagnosed Untreated Systemic 4; AL Amyloidosis 28 days long cycle Pomalidomide orally 2-4mg/day days 128; An Open-label, Phase II Study of Pomalidomide Dexamethasone orally 20-40 mg, days and Dexamethasone (PDex) for Previously 1,8,15,22 Treated Patients With AL Amyloidosis. 28 days long cycle, until progression or unacceptable toxicity Pomalidomide orally days 1-21; Phase I Study of Pomalidomide, Bortezomib, and Bortezomib iv. days 1,8,15; Dexamethasone (PVD) as First-Line Treatment Dexamethasone orally 1,8,15,22; of AL Amyloidosis or Light Chain Deposition 28 days long cycle, until progression or Disease unacceptable toxicity A Phase 3, Randomized, Controlled, Open-label, MLN9708 4mg orally days 1,8,15; Multicenter, Safety and Efficacy Study of Dexamethasone 20 mg/day orally days Dexamethasone Plus MLN9708 or Physician's 1,8,15,22; Choice of Treatment Administered to Patients 28 days long cycle With Relapsed or Refractory Systemic Light Chain (AL) Amyloidosis Pomalidomide orally 2-4mg/day, days 128; A Phase I/II Trial of Pomalidomide and Dexamethasone orally 10-20 mg, days Dexamethasone in Subjects With Previously1,8,15,22; Treated AL Amyloidosis 28 days long cycle, until progression or unacceptable toxicity

Active comparator

Condition

Estimated enrollment

Identifier

X

Untreated Systemic 54 patients NCT01807286 AL Amyloidosis

X

Primary Amyloidosis of 28 patients NCT01510613 Light Chain Type

X

Primary Systemic Amyloidosis

Physician‘s choice

Relapsed or Refractory Systemic Light Chain Amyloidosis

X

PreviouslyTreated AL Amyloidosis

36 patients NCT01728259

248 patients

NCT01659658

35 patients NCT01570387

31

A Phase II Trial of MRD (Melphalan, Lenalidomide and Dexamethasone) for Patients With AL Amyloidosis A Phase I/II Trial of Lenalidomide Combined With Cyclophosphamide and Intermediate Dose Dexamethasone in Patients With Primary (AL) Systemic Amyloidosis A Multicentric, Phase II Trial of Lenalidomide, Cyclophosphamide and Dexamethasone in Patients With Primary Systemic Amyloidosis (AL) Newly Diagnosed, Not Candidates for Hematopoietic Stem Cell Transplantation

Dexamethasone 20 mg/day orally days 1,8,15,22; Lenalidomide 10mg/day, days 1-21; Melphalan orally 5mg/m2/day days 1-4; Lenalidomide 5-25mg/day orally, days 121; Dexamethasone 20 mg/day days 1-4; Cyclophosphamide 50-100 mg/day orally days 1-10; 28 days long cycle Lenalidomide 15mg/day, day 1-21; Cyclophosphamide 300 mg/m2 days 1,8; Dexamethasone 20 mg/day orally days 14 and 9-12; 28 days long cycle, 6 cycles

X

Primary Systemic Amyloidosis

35 patients NCT00679367

X

Primary (AL) Systemic Amyloidosis

37 patients NCT00981708

X

Primary Systemic Amyloidosis (AL) Newly Diagnosed

30 patients NCT01194791

32