ALBERT

Description: Polymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes. (Blood. 2005;106:2871-2878)

Description: Abstract 3400 Poster Board III-288 To analyse the impact of complete response (CR), near CR (nCR) and very good partial response (VGPR) before and after first high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) on overall survival (OS) and progression-free survival (PFS), we evaluated all patients with multiple myeloma (MM) who underwent an ASCT in frontline treatment at our centre. The transplantations were performed between June 1992 and February 2009 giving a minimum follow up of 5 months after ASCT. The retrospective analysis included a total of 994 patients (579 males and 415 females) with a median age of 58 years at time of first ASCT (range 25 - 76 years). Median follow-up after first ASCT was 5.8 years. All patients suffered from symptomatic MM. Before induction treatment 48%, 31% and 21% of patients were in ISS-stage I, II and III, respectively. The following induction regimes were applied prior to HDT: VAD (n=683), TAD (n=74), PAD (n=64), and other regimes (n= 173). The patients were treated with HDT once (n= 460), twice (n=437) or thrice (n=97). 91 patients received an allogeneic SCT, 30 of these before first progression after ASCT. These were censored for PFS at time of allogeneic SCT. Maintenance therapy (interferon n=332, thalidomide n=203, bortezomib n=48 or others n=13) was administered in 596 patients. Overall survival and progression-free survival were calculated from the time of first ASCT. The median OS time was 5.7 years and the median PFS was 2.2 years. Log-rank test, univariate and multivariate Cox PH regression as well as landmark analyses were utilized to assess the prognostic impact of response. We analysed the effect of achievement of CR, of nCR or CR and of VGPR or CR or nCR before and after HDT, respectively. Achieving CR or nCR is a highly significant prognostic factor for PFS and OS before (p

Description: Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Background: In the HOVON65/GMMG HD4 trial in patients with newly diagnosed multiple myeloma we have previously shown that patients with renal impairment (RI) (creatinine 〉 2 mg/dl) have higher response rates and better survival when receiving bortezomib both in the induction and maintenance therapy before and after high-dose chemotherapy (HDT) (Scheid et al. Haematologica 2014). In addition patients with RI showed a higher prevalence of genetic high-risk features such as del17p or t(4;14). The aim of this analysis was to further elucidate the interaction between renal and genetic risk factors in well defined homogeneously treated myeloma patients. Methods: For this study we selected 2 cohorts of patients entered into 2 consecutive prospective trials with centralised FISH-assessement on CD138-selected bone marrow cells. The first cohort (1) comprises 395 patients from the HOVON65/GMMG HD4 trial having been treated in the German centers and the second cohort (2) consisted in the 601 patients (intention-to-treat population) from the recently closed GMMG MM5 trial. Patients lacking FISH results were excluded from the analysis, which was the case for 53 (13.4%) patients in cohort 1 and 43 (7.2%) patients in cohort 2. In cohort 1 induction treatment was vincristine or bortezomib + doxorubicin and dexamethason followed by tandem HDT followed by bortezomib or thalidomide maintenance. Cohort 2 received doxorubicin or cyclophosphamide + bortezomib and dexamethason as induction followed by 1- 2 HDT and consolidation and maintenance with lenalidomide. Results: In cohort 1 38 (10%) had RI and del 17p was found in 12/33 (36.4%) evaluable patients compared to 24/302 (7.9%) patients without RI (p 2 copies) was present in 14/33 (42.4%) patients with RI and 92/298 (30.9%) without RI (n.s.). In cohort 2 68/601 (11.3%) had RI and 7/63 (11.1%) had del17p compared to 56/495 (11.3%) patients without RI (n.s.) while 29/63 (46%) patients with RI had t(4;14) versus 265/495 (53.5%) without RI (n.s.). Gain1q21 (〉2 copies) was found in 36/63 (57.1%) with RI versus 209/495 (42.2%) without RI (p=0.025). In cohort 1 the response rate with at least VGPR after induction was low with and without RI in the VAD arm (15 vs 7.1 %) and reduced in patients with RI in the PAD arm compared to those without RI (22.2 vs 37.9%). This trend was not found in cohort 2: Patients with RI had VGPR or better in 42.4% in the PAD and 52.9% in the VCD arm, compared to 33.5% and 33.1% without RI respectively. Del17p and t(4;14) which were more frequent in patients with RI in cohort 1 did not have a negative impact on response rates after induction. Similarly gain1q was more frequent among patients in cohort 2 with RI but did not impact on response to induction. Conclusions: We analysed the effect of RI and genetic risk factors on the response to induction therapy in two different patient cohorts from two consecutive prospective trials. High-risk genetic features where found more frequently in patients with RI, but the pattern was entirely different between cohort 1 and 2 and they did not seem to influence response rates after induction. Our results confirm that bortezomib-based induction regimens achieve high response rates in myeloma patients with RI similar to those in patients without RI, independent of the presence of genetic risk factors. Disclosures Scheid: Janssen: Honoraria; Celgene: Honoraria. Salwender:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Binding site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Mai:Janssen: Travel support Other. Hose:Novartis: Research Funding. Weisel:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy, Honoraria; BMS: Consultancy; Noxxon: Consultancy. Duerig:Janssen: Consultancy, Honoraria; Celgene: Honoraria. Goldschmidt:Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Polyphor: Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Onyx: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau.

Description: Introduction Although the therapeutic armamentarium against multiple myeloma has tremendously increased in recent years, it still remains an incurable disease. A highly promising novel anti-tumoral treatment strategy is to target specific non-redundant metabolic achilles heels of individual cancer entities. The semi-essential amino acid arginine can be synthesized from citrulline in most physiological tissues due to expression of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1). Various tumor entities do not express ASS1, therefore depend on the exogenous availability of arginine and pharmacological approaches to systemically deplete arginine are in phase I-III clinical development for such arginine-auxotrophic cancers. Cell death induction by arginine depletion can be dramatically enhanced by co-application of the arginine analogue canavanine. Canavanine can be used by the respective aminoacyl tRNA synthetase instead of arginine during protein translation and this leads to a highly toxic intracellular accumulation of misfolded proteins. In preliminary work we have seen that myeloma cells are largely arginine-auxotrophic and can be killed by arginine depletion and canavanine supplementation within hours, while ASS1 expressing cells are completely protected by their endogenous arginine rescue capability. Encouraging results of tumor control have already been seen in a murine myeloma model. Methods Human myeloma cell lines (NCI-H929_A2 and FD50, developed in our laboratory) were cultured and treated in RPMI-1640 medium with or without arginine. Protein levels were determinded by western blot analysis. Cell viability was measured by propidium iodide staining and flow cytometry analysis. RNA quantification was done by qRT-PCR. For autophagosome and aggresome quantification we used immunofluorescence staining (IF) and laser scanning microscopy (LSM). Results Arginine depletion and canavanine supplementation led to misfolded protein accumulation which was followed by massive apoptotic cell death. Both processes were further enhanced by co-treatment with the proteasome inhibitor bortezomib, indicated by an increase in intracellular polyubiquitinated proteins as well as higher cleaved caspase 3 levels and propidium-iodide positive cells after only 8-12 h in both tested cell lines. Unexpectedly, the endoplasmic reticulum (ER)-stress response was activated only very moderately. Expression of CHOP, a pro-apoptotic transcription factor that is highly translated under toxic ER stress, was not altered compared to control conditions. Tunicamycin-mediated induction of enhanced ER stress significantly improved the viability of arginine-starved and canavanine treated cells. This suggests that protein accumulation mainly takes place in the cytoplasm rather than the ER and tunicamycin might alleviate cell death by reduction of total protein translation. Despite severe arginine deficiency and induction of misfolded protein stress, the cells were not able to respond by an adequate upregulation of macroautophagy, as determined by an altered LC3 metabolism. The autophagic flux was significantly reduced compared to control conditions after 4-8 h of treatment. There was a strong induction of BAG3 and p62 proteins, which are both associated with chaperone-assisted autophagy as well as aggresome formation and are normally cleared via macroautophagy. Cytoplasmic aggresome formation was not detectable until onset of apoptosis. Also, no relevant modulation of phosphorylation of the autophagy inducer mTORC and the downstream kinase p70S6K1 was noted upon arginine depletion and canavanine co-treatment. Finally, ER stress induction via tunicamycin did not improve autophagic protein turnover, as determined by IF staining, LSM and western blot. Conclusions Arginine starvation in combination with canavanine supplementation induces fast and highly efficient cell death in arginine-auxotrophic myeloma cells. This novel strategy interferes with myeloma cellular metabolism by induction of misfolded protein accumulation. A relevant upregulation of potentially protective cellular strategies like ER stress responses, aggresome formation and autophagy are either not detectable or they remain insufficient. We hypothesize that our novel metabolic anti-tumor strategy is either too potent or too fast for the tumor cells to cope with its consequences. Disclosures No relevant conflicts of interest to declare.

Description: Background The MM5 phase III trial of the German-Speaking Myeloma Multicenter Group (GMMG) was designed to address two independent primary objectives: 1.) demonstration of non-inferiority of VCD (bortezomib, cyclophosphamide, dexamethasone) induction compared to PAd (bortezomib, adriamycin, dexamethasone) induction therapy with respect to response rate (very good partial response or better). 2.) determination of the best of four treatment strategies with respect to progression-free survival (PFS). The four treatment strategies were defined by PAd vs. VCD induction treatment, high dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT) as well as consolidation and maintenance treatment with lenalidomide for 2 years vs. lenalidomide until complete response (CR). Methods 504 patients were included in the trial between July 2010 and October 2012. A non-inferiority analysis of VCD compared to PAd with respect to response rates after induction treatment with a non-inferiority margin of 10% for the difference in response rates (VGPR and better; first primary endpoint) and a safety analysis were performed. During the induction phase the patients were treated with 3 cycles of PAd or VCD. PAd was dosed as bortezomib 1.3 mg/m2, days 1, 4, 8, 11, doxorubicin 9 mg/m2, days 1-4, dexamethasone 20 mg, days 1-4, 9-12, 17-20 (repeated every 28 days). VCD consisted of bortezomib 1.3 mg/m2, days 1, 4, 8, 11, cyclophosphamide 900 mg/m2 day 1, dexamethasone 40 mg, days 1-2, 4-5, 8-9, 11-12 (repeated every 21 days). The route of administration for bortezomib was changed from intravenously to subcutaneously in all study arms by a protocol amendment in February 2012 after inclusion of 314 patients. The non-inferiority analysis was based on intention-to-treat (ITT) population (502 evaluable patients) and per-protocol (PP) population (473 evaluable patients). Responses were assessed according to the response criteria of the International Myeloma Working Group (IMWG). Results In the ITT population, patients treated with PAd or VCD were equally distributed for ISS and Durie-Salmon disease stage, LDH, kidney function and the cytogenetic abnormalities translocation t(4;14), deletion 17p13 and gain 1q21. In the PAd group, the median age of the patients was higher (59.4 vs. 58.7, p=0.04). 229 of 251 patients (91.2%) in the PAd group and 241 of 251 patients (96.0%) in the VCD group completed induction treatment. Observed response rates (PAd vs. VCD) were 4.4% vs 8.4% for complete response, 34.3% vs. 37.0% for ≥ very good partial response and 72.1% vs. 78.1% for ≥ partial response. Non-inferiority of VCD compared to PAd was shown (two-sided p=0.0026). Similar results were obtained in the PP analysis. The proportion of patients with any adverse event (AEs) was comparable in PAd vs. VCD (61.3% vs. 64.0%, p=0.58), but more serious adverse events (SAEs) were observed during PAd induction (32.7% vs. 24.0%, p=0.037). VCD led to a significantly higher proportion of leukopenia and neutropenia CTCAE grade 3 and 4 (PAd: 11.3% vs. VCD: 35.2%; p

Description: Introduction: The German-Speaking Myeloma Multicenter Group (GMMG) has initiated a randomized multicenter phase III trial on the effect of elotuzumab in VRD (bortezomib, lenalidomide, dexamethasone) induction/consolidation and lenalidomide maintenance in patients with newly diagnosed multiple myeloma (GMMG-HD6 trial, NCT02495922). The study compares four cycles induction therapy with VRD vs. VRD + elotuzumab, followed by standard intensification (i.e. mobilization and stem cell transplantation), two cycles consolidation with VRD/VRD + elotuzumab and lenalidomide maintenance +/- elotuzumab. The primary endpoint is determination of the best of four treatment strategies regarding progression-free survival. Here we present a first analysis of stem cell mobilization within this study. Patients and Methods: We performed a retrospective analysis of collection data on all patients who underwent peripheral blood stem cell (PBSC) collection between trial initiation in June 2015 and June 2016. Only patients with completely available datasets in respect of mobilization were considered (n=111). The vast majority of 99 patients (89%) received chemomobilization with CAD (cyclophosphamide, adriamycin, dexamethasone) followed by 5-10 µg G-CSF /kg body weight (bw) /d (starting day +9 until completion of PBSC collection), while in one case (1%) dexamethasone was omitted and in 10 cases (9%) cyclophosphamide mono was administered. One patient underwent steady-state mobilization with G-CSF only (10µg /kg bw /d). 55/111 patients received VRD (50%), whereas the remaining patients received VRD + elotuzumab. According to the recommendations of the study group, PBSCs for three stem cell transplants were to be collected. One transplant ideally consisted of ≥2.5 x10^6 CD34+ cells /kg bw, but in the event of poor mobilization as low as ≥2.0 x10^6 CD34+ cells /kg bw would be considered acceptable. Results: The median number of collected CD34+ cells was 10.4 x10^6 /kg bw (range 2.88 to 23.01 x10^6 /kg bw). Overall, 92 patients (83%) collected ≥7.5 x10^6 CD34+ cells /kg bw and another 12 patients (11%) collected between 6.0 and 7.5 x10^6 CD34+ cells /kg bw, resulting in three transplants, respectively. Only 7 patients (6%) collected below 6.0 x10^6 CD34+ cells /kg bw; 5 of them had been treated in the VRD-arm without elotuzumab. Due to insufficient PBSC mobilization after conventional treatment, 14 patients (13%) received a rescue mobilization with plerixafor, from which 12 patients collected ≥6.0 x10^6 CD34+ cells /kg bw. Overall, 7 serious adverse events (SAEs) occurred during mobilization phase, 4 of them in the study arm with elotuzumab. Conclusions: Cyclophosphamide-based chemomobilization after induction therapy with VRD is feasible. Efficient PBSC collection of ≥6.0 x10^6 CD34+ cells /kg bw could be performed in 104 of 111 patients (94%), with a low incidence of SAEs. The need for rescue mobilization was not higher than that of comparable previous GMMG treatment protocols. The addition of elotuzumab during induction phase did not impede PBSC collection. Disclosures Wuchter: Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bertsch:Janssen: Research Funding; Celgene: Research Funding; Chugai: Research Funding. Munder:Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Bristol Myers Squibb: Honoraria. Fenk:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding. Hillengass:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Research Funding; Sanofi: Research Funding. Raab:Novartis: Consultancy, Research Funding; BMS: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Scheid:Medac: Other: Travel, accomodations or expenses; Baxalta: Honoraria; Amgen: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel, accomodations or expenses; Janssen: Consultancy, Honoraria; Celgene: Other: Travel, accomodations or expenses; BMS: Consultancy, Honoraria. Weisel:Onyx: Consultancy; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria. Goldschmidt:Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Studies in newly diagnosed multiple myeloma (MM) described a prognostic value of the serum free light chain ratio (sFLCr) on progression free survival (PFS) and overall survival (OS). The GMMG MM5 phase III trial compares VCD (bortezomib, cyclophosphamide, dexamethasone) and PAd (bortezomib, adriamycin, dose-reduced dexamethasone) for induction therapy followed by stem cell mobilization and harvest, high-dose therapy and a lenalidomide-based consolidation/maintenance therapy for 2 years vs. lenalidomide until complete response (CR). First primary end point of the ongoing study was response after induction, second primary end point was progression-free survival. To analyse if the sFLCr has a prognostic value on the response after induction therapy with respect to achieve a very good partial response (VGPR) or better, we investigated patients with newly diagnosed MM which were included in the MM5 trial. 504 patients between 18-70 years with newly diagnosed MM were included in the MM5 trial between July 2010 and October 2012. Patient serum samples were collected and sent to a central laboratory for analysis of the free light chains (sFLC) in serum prior to treatment and after induction therapy. For the sFLC measurement the Freelite® assay (The Binding Site) was used. The analysis was based on the intention-to-treat (ITT) population (502 patients evaluable). The sFLC ratio (sFLCr) was available for n=498 patients at baseline. After induction treatment, there were 179 patients achieving CR and VGPR (VGPR+ group) and 318 patients not achieving at least VGPR (PR- group). There was no significant difference in the response between the PAd and the VCD (reported separately), so we analysed the data irrespective of the treatment arm. We examined in univariate and multivariate analyses the association of response VGPR+ after induction treatment with clinical variables (age, gender, ISS, CRAB criteria, IgA vs non-IgA type MM, WHO status), LDH measurement, cytogenetic factors (deletion 17p13, gain 1q21, translocation (4;14) ) and sFLCr at baseline. sFLCr was analysed either as categorized factor for values within the range [1/32, 32] or outside this range, as proposed by Snozek et al. (2008), or as continuous factor, where the absolute value of log2 transformed sFLCr is used for analyses. Further, LDH values were log2 transformed for analyses. In univariate analysis, the groups of VGPR+ and PR- showed no significant difference in age, gender, ISS, presence of anemia or bone disease, WHO state or the cytogenetic abnormalities del17p13, gain 1q21 or translocation t(4;14), but were significantly associated with elevated Calcium (p=0.07), renal insufficiency (p=0.004), presence of IgA type MM (p