ALBERT

Description: Key Points KRd has a favorable benefit-risk profile compared with Rd, regardless of baseline cytogenetic risk status, in patients with relapsed MM. KRd improves but does not abrogate the poor prognosis associated with high-risk cytogenetics in patients with relapsed MM.

Description: BACKGROUND: The European Commission has granted conditional approval to daratumumab (DARA) as monotherapy in adult patients (pts) with relapsed or refractory multiple myeloma (MM) whose prior therapy included a proteasome inhibitor (PI) and an immunomodulatory agent (IMiD) and who have demonstrated disease progression on the last therapy. DARA was approved under an accelerated assessment based on single-arm phase 2 studies. Outcomes in these heavily pretreated pts in a real-world (RW) setting can provide evidence on the relative treatment efficacy of DARA versus physician's choice (PC). AIMS: To perform an adjusted comparison of overall survival (OS) and progression-free survival (PFS) for DARA monotherapy versus PC, as observed in a RW historical cohort of heavily pretreated and highly refractory MM pts from the Czech Republic using pt-level data. METHODS: Using RW longitudinal pt chart data from the Registry of Monoclonal Gammopathies (RMG) of the Czech Myeloma Group, pts with ≥2 prior lines of therapy previously exposed to both a PI and an IMiD were identified. Pt-level data from the RMG were pooled from pivotal DARA monotherapy studies (pts treated with DARA 16 mg/kg). Pts in the RMG could contribute information to the analysis for multiple lines of therapy, with baseline defined as the date of initiation of the actual treatment line. For the definition of PFS, missing data for the date of disease progression for pts in the RMG who initiated subsequent therapy were replaced by the conservative proxy of the date of initiation of the next treatment. OS and PFS were analysed using a Kaplan-Meier analysis. To adjust for confounding variables, a multivariate Cox proportional hazards regression was developed that included age, gender, beta-2 microglobulin levels (β2M : 5.5 g/L), albumin levels (

Description: Background Ixazomib, the first oral proteasome inhibitor, has been approved for 〉3 years in 〉70 countries, for the treatment of RRMM pts who have received ≥1 prior therapy, on the basis of the TOURMALINE-MM1 study, which reported an overall response rate (ORR) of 78% and median progression free survival (PFS) of 20.6 mos. Although outcomes and tolerability in routine clinical practice often differ from data reported in clinical trials, growing evidence suggest that outcomes in patients treated with ixazomib-based regimens are comparable to those in the Phase 3 TOURMALINE-MM1 trial. We report on an expanded pooled analysis with longer follow-up of IRd therapy from the INSIGHT MM study (NCT02761187) and the Czech Registry of Monoclonal Gammopathies (RMG) to evaluate the effectiveness of IRd in RRMM pts in routine clinical practice. Methods INSIGHT MM is a large, prospective, observational study which has enrolled over 4,200 adult pts with MM from Europe (plus Israel, EUR), the US, Asia, and Latin America, with a planned follow-up of ≥5 years. The RMG comprises clinical data for 〉6,000 MM pts enrolled at 19 Czech and 4 Slovak centers. Eligible pts had 1-3 (INSIGHT) or ≥1 prior therapy (RMG) including an IR-based regimen. Individual pt level data on demographics, disease characteristics, treatment history, effectiveness, and safety from INSIGHT and RMG were integrated and analyzed. Best response or time to first response and PFS were determined as per investigator assessment, using IMWG criteria. PFS, duration of treatment (DOT), and overall survival (OS) were estimated using Kaplan Meier (KM) methodology, applying an exclusion criterion to account for immortal time bias (INSIGHT only). Results At data cutoff of 22 Nov 2018, 217 pts (83 in INSIGHT and 134 in RMG) from 11 countries had been included: 191 (88%) from EUR, 17 (8%) from the US, and 9 (4%) from Taiwan; 89% of pts were treated in an academic facility. At diagnosis, 32% of pts had ISS Stage III disease, 78% had bone lesions, 46% had anemia, and 12% had elevated creatinine. At study start, median age was 67 years with 12% 〉75 years; 58%/14% of pts had ECOG performance status 1/2. The distribution of immunoglobulin (Ig) heavy and light chain MM was as expected; 69% of pts had IgG MM. Overall, 21% of pts had extramedullary disease. Prior therapies included: bortezomib (90%), stem cell transplant (60%), thalidomide (47%), lenalidomide (26%), carfilzomib (8%), daratumumab (6%), and pomalidomide (2%). Median time from diagnosis to start of IRd therapy was 42.1 mos; 43%/35%/22% of pts received IRd at 2nd/3rd/≥4th line. The most common reasons for starting IRd were relapse/progression (87%) and insufficient response (10%). The most common CRAB criteria present were bone lesions (48%) and anemia (18%). Median duration of follow-up was 12.6 mos in all pts. At data cutoff, 117 (54%) pts had discontinued IRd; median DOT was 11.9 (95% CI: 9.4-15.2) mos; at 12 mos, 49% (41.3-56.2) of pts were still on treatment (KM estimates). Data on best response to therapy were available for 152 pts. The ORR was 74%, with 36% ≥VGPR; ORR with IRd at 2nd/3rd/ ≥4th-line therapy was 82%/71%/59%, including 43%/37%/17% ≥VGPR. Median time to first response was 1.2 mos (RMG); median time to best response was 3.7 mos (INSIGHT). Median PFS was 21.6 (95% CI: 13.6-26.7) mos across all lines. PFS rate at 12 mos was 62%, and 86 (40%) pts had progressed at data cutoff. Median time to next therapy (TTNT) was 31.5 (95% CI: 24.5-35.9) mos, with a 12-month rate of 74% across all lines. Overall, 60 (28%) pts received subsequent therapies including daratumumab (22%), pomalidomide (22%), bortezomib (20%), carfilzomib (17%), lenalidomide (15%), and thalidomide (12%). At data cutoff, 53 (24%) pts had died. Median OS was 36.7 (95% CI: 24.4-NR) mos, with 79% of pts alive at 12 mos (Figure). Regarding safety, ixazomib and lenalidomide dose reductions were required in 16% and 36% of pts, respectively, including 10% and 21% who required dose reductions due to AEs. Conclusions These findings show that the effectiveness of IRd in routine clinical practice (ORR 74%, median PFS 21.6 mos) is comparable to the efficacy of IRd reported in the registrational TOURMALINE MM1 trial (ORR 78%, median PFS 20.6 mos). IRd is well tolerated with no new safety signals, and low rates of dose reductions due to AEs for ixazomib (10%) and lenalidomide (21%). Outcomes should be interpreted with caution due to limited maturity of data. Disclosures Hajek: Janssen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Amgen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Celgene: Honoraria, Other: Consultant or advisory relationship, Research Funding; AbbVie: Other: Consultant or advisory relationship; Bristol-Myers Squibb: Honoraria, Other: Consultant or advisory relationship, Research Funding; Novartis: Other: Consultant or advisory relationship, Research Funding; PharmaMar: Honoraria, Other: Consultant or advisory relationship; Takeda: Honoraria, Other: Consultant or advisory relationship, Research Funding. Minarik:Celgene: Consultancy, Honoraria, Research Funding; Amgen, BMS, Janssen-Cilag, Takeda: Consultancy, Honoraria. Straub:Amgen, Takeda, Celgene: Consultancy. Berdeja:Amgen Inc, BioClinica, Celgene Corporation, CRISPR Therapeutics, Bristol-Myers Squibb Company, Janssen Biotech Inc, Karyopharm Therapeutics, Kite Pharma Inc, Prothena, Servier, Takeda Oncology: Consultancy; AbbVie Inc, Amgen Inc, Acetylon Pharmaceuticals Inc, Bluebird Bio, Bristol-Myers Squibb Company, Celgene Corporation, Constellation Pharma, Curis Inc, Genentech, Glenmark Pharmaceuticals, Janssen Biotech Inc, Kesios Therapeutics, Lilly, Novartis, Poseida: Research Funding; Poseida: Research Funding. Boccadoro:AbbVie: Honoraria; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Spencer:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Secura Bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Specialised Therapeutics Australia: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. van Rhee:Karyopharm Therapeutics: Consultancy; Kite Pharma: Consultancy; Adicet Bio: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy. Thompson:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; VIA Oncology: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties: Myeloma reviewer; GSK: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Doximity: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; BMS: Research Funding; Lynx Bio: Research Funding. Abonour:BMS: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Chari:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncoceutics: Research Funding; Novartis Pharmaceuticals: Research Funding; GlaxoSmithKline: Research Funding; Array Biopharma: Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy; Pharmacyclics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Cook:Karyopharm: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Costello:Takeda: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Davies:Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor; Janssen, Celgene: Other: Research Grant, Research Funding. Hungria:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lee:Amgen: Consultancy, Research Funding; GlaxoSmithKline plc: Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Leleu:Sanofi: Honoraria; Takeda: Honoraria; Oncopeptide: Honoraria; Karyopharm: Honoraria; Amgen: Honoraria; Carsgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Merck: Honoraria. Puig:Takeda: Consultancy, Honoraria; The Binding Site: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Rifkin:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Terpos:Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding. Usmani:Bristol-Myers Squibb: Consultancy, Research Funding; Sanofi: Patents & Royalties, Research Funding, Speakers Bureau; Janssen: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Takeda: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Celgene: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding; Pharmacyclics: Patents & Royalties, Research Funding; Amgen: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Array Biopharma: Patents & Royalties, Research Funding; Skyline DX: Consultancy. Weisel:Sanofi: Consultancy, Honoraria, Research Funding; Juno: Consultancy; GSK: Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Research Funding; Adaptive Biotech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Zonder:Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Intellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Caelum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alnylam: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees. Skacel:Millennium Pharmaceuticals, Inc., subsidiary of Takeda Pharmaceutical Company Limited: Employment. Elliott:Takeda: Employment. Demers:Takeda: Employment. Stull:Takeda: Employment. Ren:Takeda: Employment. Maisnar:Janssen, Amgen, Celgene, Takeda, BMS: Consultancy, Honoraria.

Description: Background: Primary mediastinal large B-cell lymphoma (PMBCL) is a rare subtype of diffuse large B-cell lymphoma with typical clinical, pathological and genetic features. No standard frontline protocol is widely accepted and role of adjuvant radiotherapy is not established yet. New intensive etoposide-based (DA-EPOCH; Dunleavy K, NEJM 2013) regimens with added rituximab show survival benefit but certain patients are still at risk of early treatment failure. As residual masses (CT) after therapy are frequent, assessment of tumor metabolic response is of utmost importance. Complete metabolic response (CMR) measured by positron emission tomography (PET) has high predictive value after intensive treatment (Martelli M, JCO 2014), but the role of interim scans is unclear. Aim: To analyze the prognostic impact of interim and final whole-body PET-CT fusion scans using visual and Deauville criteria in PMBCL patients receiving intensive etoposide-based regimens with rituximab. Patients: Thirty-four consecutive PMBCL patients were treated in a single center from 5/2005 to 6/2013. The median age at diagnosis was 32.5 (19-73) years; male-to-female ratio 0.62:1. Ann Arbor stages I through IV were observed in 1, 19, 3 and 11 patients, respectively. Large mediastinal mass (≥11cm) was present in 19 (56%) patients. Thirteen patients (38%) had concurrent extranodal disease but unaffected bone marrow. IPI and age-adjusted IPI scores were: low 16 (47%) and 8 (24%), low-intermediate 11 (32%) and 11 (32%), high-intermediate 5 (15%) and 12 (35%), and high 2 (6%) and 3 (9%) patients, respectively. All patients received rituximab; intensive etoposide-doxorubicin-based therapy was given to 30 (88%; sequential n=29, MegaCHOP-ESHAP n=1), intensified CHOP (PACEBO) to 1 and CHOP to 3 patients. Treatment was consolidated with ASCT (BEAM 200) in high-risk bulky cases with extranodal involvement (n=20, 59%). Involved-field radiotherapy was used in only 9 (26%) patients. Whole-body PET-CT scans were planned after 2nd chemotherapy cycle (interim; iPET-2) and treatment completion (final; fPET). The PET results were expressed as positive/negative (IHP; Juweid ME, JCO 2007) and 5-point Deauville scale (D1-5; Meignan M, Leuk Lymphoma 2014). Treatment response was assessed using revised criteria (Cheson BD, JCO 2007). Results: After treatment, 28 (82%) patients achieved complete remission (CR), one partial remission, three stable disease and two progressed. Seventeen CR patients (61%) had residual mass on CT (median longest diameter 40mm). After a median follow-up of 58.7 months, 8 (24%) patients relapsed or progressed and 6 (18%) of them died. Five-year overall survival (5-y OS) reached 81.2% (95% CI 0.68-0.95), 5-year progression survival (5-y PFS) was 75.5% (95% CI 0.61-0.90). There were 27 (79%) and 33 (97%) cases assessable with iPET-2 and fPET, respectively. Using visual criteria, iPET-2 was negative in 10/27 (37%) and fPET in 28/33 (85%) cases. With Deauville criteria, iPET-2 scores were D1-2 in 6 (22%), D3 in 4 (15%) and D4-5 in 17 (63%) cases, and fPET scores D1-2 in 19 (58%), D3 in 8 (24%) and D4-5 in 6 (18%). All visually negative iPET-2 were scored as D1-3. In fPET, there was only one discordant case (visually negative scored as D4). Visually negative (D1-3) iPET-2 was associated with superior 5-y OS (34% vs 100%, p=0.08) and 5-y PFS (65% vs 100%, p=0.049). Visually negative visual fPET was linked with superior 5-y OS (20% vs 96.0%, p

Description: Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens. Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2. Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%). Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A). Supported by the grant NT 14393. Disclosures No relevant conflicts of interest to declare.

Description: Background: Myeloma bone disease (MBD) is present in up to 80% patients with newly diagnosed multiple myeloma (MM). It is known to be activated by three separate signaling pathways - receptor activator of nuclear factor κB/osteoprotegerin pathway (RANK/OPG), macrophage inflammatory factor pathway (MIP) and Wnt pathway. Except of these, several other factors have been described in relationship to MBD development. The aim of our study was to assess the activity of selected parameters of MBD signaling with respect to macroscopic skeletal changes detectable using imaging techniques, and in comparison with markers of bone turnover and the activity of MM. Patients and methods: We assessed 92 patients with monoclonal gammopathies: 56 patients with active multiple myeloma, 12 with smoldering myeloma and 24 individuals with monoclonal gammopathy of undetermined significance (MGUS). Following parameters of MBD signaling were measured in the patients´serum: hepatocyte growth factor (HGF, Human HGF Quantine ELISA), macrophage inflammatory factor alpha (MIP-1α, Human CCL3/MIP-1α Quantikine ELISA), syndecan-1 (Human Syndecan-1 (CD138) ELISA), osteoprotegerin (OPG, Human Osteoprotegerin ELISA), Activin A (Human Activin A Quantikine ELISA), Dickkopf-related protein-1 (DKK1, Human Dkk-1 Quantikine ELISA), Annexin A2 (Human Annexin A2 ELISA kit), nuclear factor kappa B (NF-κB, Human Nuclear factor-kappa B). The extent of skeletal involvement was evaluated using whole body magnetic resonance and low-dose computed tomography. The levels of the selected parameters of MBD signaling were compared to serum levels of Ca, P, PINP, ICTP, CTx, parathormone (PTH), calcitonine (CLC), 25-hydroxyvitamin D2 (D2), 1,25dihydroxyvitamin D3 (D3), bone isoenzyme of alkaline phosphatase (bALP) and sclerostin (SCL). The parameters were measured at the time of diagnosis and then after 4 months. In patients with MM we measured selected parameters of MBD in comparison with the stage of the disease according to Durie-Salmon (DS) and according to International Staging System (ISS). In MGUS we assessed the relationship of the parameters to the risk of transformation to MM based on the Mayo clinic stratification system. For statistical estimation we used Kruskal-Wallis test and Mann-Whitney U post hoc test with Bonferroni correction at p 〈 0,05. Results: Statistically significant difference between patients with low and high skeletal involvement was found in serum levels of MIP-1α, syndecan-1, DKK-1 and Annexin A2. None of the parameters correlated with the presence of extramedullary disease. With respect to markers of bone turnover we found following significant relationships: HGF to PINP, ICTP, CTx and inverse relationship to D3; MIP-1α to PINP, ICTP, CTx and inverse relationship to D3; Syndecan-1 to ICTP, CTx, and inversely to D2 and D3; OPG to ICTP and SCL; Activin A to PINP, ICTP, CTx and SCL; NF-κB to ICTP and CTx. We found significant differences between MGUS and MM in serum levels of HGF, MIP-1α, syndecan-1, DKK-1 and NF-κB. There was no significant relationship of any of the parameters to DS, however, we found significant relationship of HGF, syndecan-1, OPG and Activin A to ISS. MIP-1α only had a significant relationship to the risk stratification of MGUS. There was no significant change in the parameters in MM patients after 4 months of treatment with respect to treatment response. Conclusions: We confirmed the relationship of MBD signaling to the extent of skeletal changes using imaging techniques. All three signalling pathways were involved in the development of MBD in MM in comparison with MGUS. Signaling involved in osteoclast activation was more activated than the parameters preventing osteoblast differentiation. There was, however, no significant change in any of the parameters in MM patients with respect to treatment response after 4 months, suggesting that bone reparation processes are delayed after hematological response. Significant relationship to the markers of bone turnover and to the activity of MM suggests possible use of the parameters of MBD signaling as tools for early detection of bone marrow changes and timely indication for anti-resorption therapy. Supported by the grant NT 14393. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5027 Background: Novel agents, such as immunomodulatory drugs and proteasome inhibitors, have substantially changed the treatment paradigm of multiple myeloma (MM). Until now, many combination regimens with novel drugs have been published, but different regimens have rarely been compared in the treatment of the first relapse of MM. Aims: In this report, we describe the outcome of a cohort of 209 patients (pts) with the first relapse of MM treated with thalidomide-based and bortezomib-based regimens with aims to compare the efficacy of these therapies. Methods: Eighty-two percent of pts (171/209) had stage III according to Durie-Salmon (DS), 17% (35/209) II and 1% (3/209) I, clinical stages according to International Staging System (ISS) were the following: stage 1 in 46% of pts (92/199), stage 2 in 29% of pts (57/199) and stage 3 in 25% of pts (50/199). Renal insufficiency was presented in 14% of pts (30/209). Median age was 64 years (range: 34–84). Thalidomide-based (T) regimens were used in 105 pts; thalidomide + alkylating agent + dexamethasone: 91 cases (87%), thalidomide + dexamethasone: 5 cases (5%), alone thalidomide: 9 cases (8%). The daily dose of thalidomide was 100–200 mg. Bortezomib-based (B) regimens were used in 106 pts; bortezomib + alkylating agent + dexamethasone: 58 cases (55%), bortezomib+alkylating agent or dexamethasone: 40 cases (38%), alone bortezomib: 8 cases (7%). Bortezomib was used in standard dose 1.3 mg/m2 intravenously on days 1, 4, 8, 15. The cycle repeated on day 21–28 for up to 8 cycles or until progression. Median of T and B treament cycles was 5, range 1–8. No statistically significant differences were observed between the T and B groups in baseline clinical parameters including age, clinical stages according to ISS and DS staging systems, the type of M-protein and presence of renal impairment. The first-line therapy was comparable for both T and B groups and it is not include novel agents. Median follow-up from start of treatment was 19.5 months (range 0.9–53.3). Results: In T group, overall response rate (ORR) was 51% (51/100), 11% of pts (11/100) achieved the complete response (CR), 16% of pts (16/100) were in very good partial response (VGPR), 24% of pts (24/100) in partial response (PR), 9% of pts (9/100) had minimal response (MR) or stable disease (SD) and 40% of pts (40/100) had progression of disease. Median time to progression (TTP) from the start of relapse treatment was 13.1 months, median overall survival (OS) was 30.4 months. Patients in T group with ORR had significantly longer OS than pts without ORR (median 46.1 months versus 22.1 months; p = 0.005). Clinical stages according to ISS in T group significantly influenced both TTP (p = 0.004) and OS (p = 0.001). In B group, ORR was 50% (34/68), 9% of pts (6/68) were in CR, 16% of pts (11/68) were in VGPR, 25% of pts (17/68) in PR, 17% of pts (12/68) had MR or SD and 32% of pts (22/68) had progression of disease. Median TTP from start of relapse treatment was 16.7 months, median OS was 37.2 months. Patients in B group with ORR had significantly longer OS than pts without ORR (median 50.1 months versus 21.2 months; p = 0.002). Clinical stages according to ISS in B group did not significantly influence TTP (p = 0.483) and OS (p = 0.207). Age, renal impairment and type of M-protein did not significantly affect TTP and OS in both T and B groups. Conclusion: The thalidomide-based and bortezomib-based regimens are similarly effective in treatment of the first relapse MM with ORR 50–51%. No statistically significance differences were observed between thalidomide-based and bortezomib-based regimens in terms of TTP (p = 0.207) and OS from the start of relapse treatment (p = 0.889). Disclosures: No relevant conflicts of interest to declare.

Description: Aim: Myeloma bone disease (MBD) is present in 80-90% patients with multiple myeloma (MM). Up to now, three signalling pathways have been described in the pathogenesis of MBD – receptor activator of nuclear factor kappa B and its ligand (RANK/RANKL) pathway, macrophage inflammatory proteins (MIP) pathway, and wingless type (Wnt) pathway. Moreover, several cytokines and parameters of bone microenvironment have been shown to interfere with bone homeostasis in MM. The aim of our study was to assess the activity of selected parameters in bone marrow of patients with MM and monoclonal gammopathy of undetermined significance (MGUS) in order to define the principal processess occuring within MBD. Materials and methods: We designed a prospective study aimed at signalling in MBD. Formaline-fixed, parafin-embedded diagnostic tissue of patients with MM and MGUS has been processed in routine tissue sections (app. 5 um) and placed on plus-charged slides. After the antigen retrieval (Table 1) indirect immunohistochemical evaluation has been processed with the use of commercial available primary antibody for particular detected protein (according to manufactor´s manual) in optimalised dilution. For the visualisation secondary antibody has been applicated with the use of the standard method avidin-biotin (ABC). Following parameters have been evaluated: RANK on myeloma and mononuclear stromal cells, RANKL and osteoprotegerin (OPG) on stromal cells, MIP-1α in plasma cells (both membrane and cytoplasm), sclerostin, MMP 9 and DKK-1 in the cytoplasm of plasma cells, Annexin A2 in plasma and stromal cells, tartrate resistant acid phosphatase (TRAP) in the cytoplasm of osteoclasts and precursor cells, Activin A in the nucleus of plasma cells, p50, p52, p62, p65, in nucleus and cytoplasm of plasma cells. Results: Activity of RANK varied between 0-100% in plasma cells with 0% activity in stromal cells. Positivity of RANKL was found on endosteum of stromal cells in 12/17 patients (71%). The activity of OPG on stromal cells was in all patients under 10%. Assessment of MIP-1α revealed 100% positivity in 9/17 patients (53%), in 13 patients (76%) the activity was more than 50%. The activity of sclerostin reached 90-100% in all patients. The levels of DKK reached in 3 patients more than 60%, in the rest it was under 10%. The levels of Annexin A2 in stromal cells were low, in 16/17 patients below 20%. In plasma cells, higher activity (above 60%) was found in 4 patients (24%). Activity of p50 above 70% was found in cytoplasm of 2 patients only (12%). The levels of p52 varied between 1-90%, majority of the patients (53%) having more than 80% activity. Similar results were found within the assessment of p65. The levels of p62 were with high activity above 70% in 16/17 patients. The activity of MMP-9 was in 9/17 patients above 70%, the rest of patients had the activity of MMP-9 under 20%. Conclusions: Patientswith monoclonal gammopathies displayed significant activation of all three signalling pathways of MBD. There were however, differences in the involvement of each individual pathway as well as in the levels of other cytokines participating on bone homeostasis, suggesting different mechanisms of the cascade occurring in patients with similar skeletal involvement. With support of the grant NT14393. Abstract 5679. Table 1 Primary antibodies Antibodies clone Antigen retrieval Dilution Source Anti- MIP1alfa C-16 MW 1:200 Santa Cruz Anti- RANK 9A725 MW 1:100 Santa Cruz Anti- RANKL N-19 MW 1:100 Santa Cruz Anti- Ann A2 Polyclonal MW 1:1000 Abcam Anti- TRAP Polyclonal MW 1:1000 GeneTex Anti- Act A Polyclonal MW 1:50 Sigma-Aldrich Anti- OPG N-20 MW 1:50 Santa Cruz Anti- p50 E-10 MW 1:50 Santa Cruz Anti- p52 C-5 MW 1:100 Santa Cruz Anti- p65 F-6 MW 1:100 Santa Cruz Anti- p62 SQSTM1 (ab56416) MW and methanol unblocking 1:50 Abcam Anti- Sclerotisin Polyclonal MW 1:100 Abcam Anti- MMP9 Polyclonal MW 1:50 Abcam Anti- Dkk-1 H-120 MW 1:100 Santa Cruz Abbreviations: MW – microwave oven Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Adding a methyl group on the cytosine residues of CpG dinucleotides (CpGs) is a basic phenomenon in DNA methylation. This modification belongs to a group of epigenetic changes that are thought to play one of the key roles in tumor development and progression. DNA hypermethylation occurs mainly in CpG islands of the promoter region in tumor-supressor (TS) genes, which leads to gene silencing. On the other hand, global DNA hypomethylation leads to a genomic instability. Many specific genes are epigenetically changed in individual malignant diseases and these genes are intensively studied by the scientific community. According to the recent research in plasma cell neoplasms, the global genic hypomethylation is predominant in contrast to hypermethylation events. Epigenetic changes of TS genes may worsen prognosis, drug response and microenvironment interaction in multiple myeloma patients. Our aim was to assess the methylation level of the RIL and p15INK4b gene. RIL protein acts as suppressor of cell proliferation and it sensitizes tumor cells to apoptosis, on the other hand, the p15 INK4bplays an important role in inhibiting the cell cycle progression in G1 phase. Methods: Our updated study includes 72 bone marrow aspirate-samples from 68 patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS). 68 samples were acquired at the time of the diagnosis and four patients with MM were assessed also in remission after chemotherapy. Extracted DNA after bisulfite modification was examined by pyrosequencing method (Pyromark Q96, Qiagen, Germany) and the selected gene regions – RIL promotor, p15INK4b promotor and p15 INK4b exon were analysed. Nine CpGs in the RIL promoter, thirteen CpGs in the p15INK4b promoter and sixteen CpGs in the p15INK4bexon were studied. Average methylation level (MtL) was generated and samples were sorted according to the mean MtL (%) into four groups for the RIL gene (less than 10% MtL, 11-19% MtL, 21-50% and more than 50%). We used commercially available unmethylated DNA and methylated DNA control. Statistical analysis was performed using Pyromark Software and Wilcoxon rank sum test. Results: Out of the four groups for the methylation level of the RIL gene promoter, the groups with 11-19% MtL and 21-50% MtL, detected significant and highly significant differences respectively at both p-values - p-value ≤0.05 and p-value ≤0.01. There were only 2 samples with MtL level over 50% in patients with active MM, precluding valid statistical analysis. In both regions of the p15 INK4b gene, i.e. the promoter and the exon, we did not find any significant differences between the methylation levels - mean MtL ranged from 3% to 6% in p15INK4b promotor and from 3% to 12% in p15 INK4b exon. The time-dependend analysis of the DNA methylation level changes in four patients assessed at the time of diagnosis and in remission of the disease after chemotherapy course revealed a significant decrease of CpGs methylation in all MM patients reaching therapeutic response. Conclusions: Updated data on the epigenetic analysis in patients with monoclonal gammopathies confirmed an important role of the RIL gene in the epigenetic network acting in pathogenesis of MM. The presented results suggest possible prognostic value and therapeutic target in patients with MM. In contrast, the p15 INK4b gene known as a frequent focus of epigenetic modifications in many tumors, did not show statistically significant differences in our study. Our results indicate that for DNA methylation analyses of MM or MGUS patients, the RIL gene could be more useful marker than the p15 INK4b gene. Moreover, the decrease of the methylation level in patients undergoing systemic chemotherapy might be associated with treatment response, suggesting its potential prognostic value. The paper was supported by the grant IGA MZ CR NT14393. Disclosures No relevant conflicts of interest to declare.

Description: Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer. Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used. Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation. Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a. With support of the grant NT14393. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Disclosures No relevant conflicts of interest to declare.