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The authors studied the influence of a six hour infusion of 25 I.U. of ACTH on the contractility and motility of leukocytes and on the absolute number of neutrophils in 10 convalescent patients. The findings were as follows: 1. A significant increase occurred in leukocyte motility after the ACTH infusion, together with an increase in contractility. 2. The authors assume that leukocytosis following ACTH is due to an increase in the contractility of neutrophil leukocytes. 3. In view of the differences in the bone marrow reserve of mature neutrophils, it is impossible to demonstrate a direct correlation between the increase in motility, contractility and the absolute number of neutrophils. 4. An indirect estimate of the bone marrow granulocyte reserve can be made from the absolute number of neutrophils and the degree of leukocyte motility.

Hajek: BMS: Honoraria; Onyx: Consultancy; Novartis: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Introduction:Release of the aberrant plasma cells (PC) from the bone marrow (BM) and their presence in the peripheral blood (PB) is a maker of disease progression and worse survival in multiple myeloma (MM) (Nowakowski et al., 2005). Circulating plasma cells (cPCs) are able to survive without homing microenvironment, evade the original tumor and colonize other bone marrow niche. Detailed analysis of various surface proteins showed that cPCs display decreased levels of integrins, adhesion molecules N-CAM (CD56) and the stem cell factor receptor (Paiva et al., 2013). Comprehensive analysis of the genome-wide gene expression profiling that could provide deeper insight into the expression patterns of cPCs of MM is still lacking.

Hajek: Janssen-Cilag: Honoraria; Celgene, Amgen: Consultancy, Honoraria.

Background:Multiple myeloma (MM) is a lymphoproliferative disease characterized by the clonal expansion of neoplastic plasma cells within the bone marrow. The genome of the malignant plasma cells is extremely unstable characterized by a complex combination of structure and numerical abnormalities. DNA copy number variants (CNV) affects target gene expression but such affectation is not compulsory and target gene expression can be modulated. We supposed that this modulation serves as a compensatory mechanism to keep genomic homeostasis. Further compensation mechanisms exhausting leads disease aggressiveness.

Palumbo: Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria.

Hajek: Merck: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria.

Background

Background

In multiple myeloma (MM), unlike normal plasma cells (PC), myeloma cells retain the self-renewing potential. Majority of medullary myeloma cells regardless over-expression of cyclins D stay in the G1 phase due to pro-apoptotic and cell cycle regulatory capacity of p53 depended axis. Nevertheless, after leukemic transformation in secondary plasma cell leukemia (PCL) or de novo in case of primary PCL, bone marrow myeloma cells become highly proliferative and even presenting as circulating plasma cells in the peripheral blood. We anticipate that complex “re-setting” of cell cycle gene coordination during leukemic transformation creates required background to restore proliferation activity and breakthrough mitotic restriction points.The objective of our study was to define and describe complex “re-setting” of cell cycle gene coordination in MM and PCL.In total, 7 healthy donors, 6 multiple myeloma and 7 plasma cell leukemia samples enrolled in this study. The mRNA from CD138+ cells was isolated using uMACS mRNA Isolation Kit Small scale (Miltenyi Biotech) and directly amplified and labeled using WT-Ovation™ Pico RNA Amplification System Version 1.0 plus WT-Ovation™ Exon Module Version 1.0 (NuGEN). Generated SPIA-cDNA was fragmented & labeled using Encore™ Biotin Module (NuGEN). cDNA was hybridized to Affymetrix GeneChip Human Exon 1.0 ST Arrays (Affymetrix, Santa Clara, USA). All samples were labeled and scanned in a randomized order to avoid batch effects. Gen sets, connected with cell cycle regulation (GO:0045786; GO:0045787) with all direct descendants (child terms) and regulation of apoptotic process (GO:0043065; GO:0043066), were taken for the Gene Set Enrichment Analysis (GSEA) and Gene Set Differential Coordination Analysis (GSDCA).Comparing of PCL, MM and healthy donors revealed coordinating changes between regulation of mitosis (GO:0045839; GO:0045840), apoptosis (GO:0043065; GO:0043066) and cell cycle arrest (GO:0007050). These changes were relevant for both positive and negative regulation sets. Gene expression profiling of MM samples revealed affected early phases of cell cycle (G1 phase and G1/S transition). In PCL samples co-expression changes was associated with late phases of cell cycle (G2/M transition, S and M phase) together with severe alteration in early phases. The mechanisms controlling differential cell cycle coordination were based on bioinformatic analysis suggested to include alternative transcription start sites, exon skipping and shortening of 3'UTR. The probe sets covering the 3'UTR of CCND2 were for example significantly down regulated in plasma cells of MM and PCL as compared to healthy donors supporting the existence of a phenomenon observed in breast cancer where shortening of 3'UTR mRNA CCND2 confers higher mRNA stability leading to higher protein expression and more cells to enter the S phase.Considering revealed coordination changes allow us to offer following statements. Expression of cell cycle positive regulators is in dynamic equilibrium with cell cycle negative regulators. We suppose that this equilibrium serves as a compensatory mechanism to oncogenic events. Despite compensation mechanisms activation, whole regulatory complex seems to be imbalanced by growing “oncogenic stress” during MM to PCL progression.This study was supported by grants NT11154, NT12130, NT13190 and the EU 6th FP to MSCNET (LSHC-CT-2006-037602), the Danish Cancer Society, the Danish Research Agency (#2101-07-0007) and the KE Jensen Foundation (2006-2008).No relevant conflicts of interest to declare.

In multiple myeloma (MM), unlike normal plasma cells (PC), myeloma cells retain the self-renewing potential. Majority of medullary myeloma cells regardless over-expression of cyclins D stay in the G1 phase due to pro-apoptotic and cell cycle regulatory capacity of p53 depended axis. Nevertheless, after leukemic transformation in secondary plasma cell leukemia (PCL) or de novoin case of primary PCL, bone marrow myeloma cells become highly proliferative and even presenting as circulating plasma cells in the peripheral blood. We anticipate that complex “re-setting” of cell cycle gene coordination during leukemic transformation creates required background to restore proliferation activity and breakthrough mitotic restriction points.

Multiple myeloma (MM) is a malignancy of differentiated B-lymphocytes characterized by accumulation of clonal plasma cells (PCs) in the bone marrow. Genome of malignant PCs is extremely unstable and characterized by complex combination of structural and numerical abnormalities. The basis of genomic instability underlying MM is still unclear. Centrosome amplification (CA) is present in about a third of MM cases and may represent a mechanism leading to genomic instability in myeloma. We have previously shown that CA is present in pre-plasma cells stages of B-cell development.The objective of our study was to evaluate changes in expression of chosen key genes involved in centrosome duplication process in both B and PCs, including their possible changes during MM progression.In total, 56 patients were evaluated for this study. Patients' characteristics were as follows: males/females 38/37, median age 69 years (range 43–83 years). Newly diagnosed (27/56) and relapsed (29/56) patients were included in this study; most of them had advanced stage of MM (DS III n=48; ISS II/III n=45). qRT-PCR (51 PCs and 32 B cell samples) was performed on a chosen set of mitotic genes, according to their role in normal centrosome duplication process.Expression of genes involving in centrosome duplication process was studied in B cells of MM patients and healthy donors. Group of MM patients showed significant increase in relative quantification coefficient R in the following genes: AURKA, AURKB, CCNB1, CCNB2, HMMR, PLK4, TACC3 and TUBG1. It is notable that TUBG1 was approximately 330-times overexpressed in MM B cells vs healthy donors.Expression of studied genes was significantly different in B and PCs populations of MM patients: AURKB and TACC3 were upregulated in B cells while CENT2 and TUBG1 were upregulated in PCs.Analyses of gene expression in newly diagnosed and relapsed patients showed significant changes of the following genes: AURKA and TACC3 were 2-times upregulated in PCs of relapsed patients; no differences were detected in population of B cells.Considering revealed changes in B cells of myeloma patients, we suspect early damage in cell-cycle regulating mechanism. This hypothesis correlates with our previous findings of CA in MM B cells. We anticipate that dynamic accumulation of showed changes plays a role in final transformation of malignant PCs. We assume that centrosomal disruption likely contributes to accumulation of genomic abnormalities in tumor cells during MM progression which can be proven in future long-term study.This study was supported by grants NT11154, NS10207, MSM0021622434No relevant conflicts of interest to declare.

Abstract 1806

Increased bone marrow angiogenesis is a characteristic feature of multiple myeloma (MM). New blood vessels formation is provided by recruiting vascular endothelial cells from existing capillaries or circulating endothelial progenitor cells (EPC). EPCs have distinct monocytic features and can be cultured from CD14+ cells. In addition, monocytes were shown to contribute to angiogenesis as EPCs in vivo. It has been established that myeloid progenitor cells participate in the development of endothelial precursors. In MM, it has been reported that circulating EPCs carried the same chromosomal aberrations as neoplastic plasma cells (PC). It is possible that EPCs originate from common precursor that gives rise to both PCs and endothelial cells. The purpose of our pilot study was to clarify whether myeloid cells, CD14+ monocytes (CD14+MO), in MM might carry the same chromosomal abnormalities as CD138+PC.Total of 15 MM patients were enrolled in this pilot study; 93 % (14/15) typical MM; 7 % (1/15) plasma cell leukemia (PCL). CD138+PCs were isolated by magnetic-activated cell separation (MACS) from bone marrow mononuclear cells (BMMNC). CD14+MOs were isolated by fluorescence-activated cell sorting (FACS) from BMMNC after isolation of CD138+PCs. The purity of both cell populations was > 90%. Distinct BM cell fractions were analyzed for del(13q14), del(17p53), IgH gene rearrangement, hyperdiploidy and trisomy of chromosomes 5, 9, 15 by interphase fluorescence in situ hybridization (FISH) according to standard protocol. The cut-off level for detection of choromosomal aberarations was set to 20%.FISH analysis proved at least one of studied chromosomal abnormalities in 20% (3/15) of MM patients (See Table 1; No.1 – No.3). In case No.1, a patient with PCL, CD14+MOs were positive for del(13q14)(31%), IgH gene rearrangement (46%) and trisomy 9 (47%). The same aberrations were found in CD138+PCs but with higher frequency (Table 1). Patient No.2 was positive for del(13q14)(35%) and for del(17p53)(39%) in CD14+MO but in CD138+PCs, the patient was positive further for hyperdiploidy (53%) and trisomy 5 (28%), 9 (56%) and 15 (60%). CD14+MOs of patient No.3 were positive only for trisomy 15 (25%) which corresponded to trisomy 15 (24%) in CD138+PCs. Other chromosomal abnormalities found in CD138+PCs of No.3 were not detected in CD14+MOs. Chromosomal aberrations identified in CD14+MOs of No.4 – No.15 did not reach the cut-off level for positivity (Table 1).In our pilot study, we demonstrated for the first time that bone marrow CD14+ monocytes of MM patients carried the same chromosomal aberrations as CD138+ plasma cells. The possible explanation is that CD14+MOs and CD138+PCs may origin from a common precursor. We hypothesize that myeloid cells might play a role in blood vessel formation as endothelial precursors and might be important in pathogenesis of multiple myeloma. It requires further study to elucidate the relationship between two distinct bone marrow populations and a potential role of CD14+cells in disease severity. Supported with research program MSM of Czech republic Nr. 0021622434 and LC06027.No relevant conflicts of interest to declare.

Centrosome aberrations are common in many types of human malignancies and are associated with aneuploidy. Loss of centrosome duplication control will often create multipolar spindles that in turn could be responsible for incorrect segregation of whole chromosomes leading to aneuploidy. Hyperdiploidy (subtype of aneuploidy) is one of the most frequent cytogenetic abnormalities in multiple myeloma (MM), where molecular changes are among the primary genetic events in disease pathogenesis. But no correlation between centrosome aberrations and aneuploidy in MM has ever been found.The objective of our study was to evaluate association of MM ploidy category with centrosome amplification in both B and plasma cells subpopulations and to investigate structural defects (gain/loss) and gene expression changes in genes controlling centrosome numbers.Immunofluorescent labeling was used for evaluation of centrosome amplification (CA) in B-cells (CD19+) and PCs (CD138+) of MM patients. Centrin (centrosome protein) copy numbers were used to define presence of centrosome amplification (CA) in cells: cells with more than 4 signals of centrin were considered to be positive. Samples with ≥11% of B-cells or ≥10% of PCs with >4 fluorescence signals of centrin were considered as CA positive. A total of 140 patients were evaluated for CA in PCs and/or B-cells, including 50 patients where both cell types were analyzed. The patient population characteristics were as follows: males/females 67/73, median age of 66 years (range, 40–92 years). Newly diagnosed (52/140) and relapsed (88/140) patients were included in this study; most of them had advanced stage of MM (DS II/III n = 107; ISS II/III n = 92). Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH), oligonucleotide-based arrayCGH (20 patients) and qRT-PCR (5 CA positive vs 5 CA negative patients) were performed on plasma cells. Hyperdiploidy analysis was done using Multi-Color Probe Panel (LSI D5S23/D5S721, CEP 9 and CEP 15) for chromosome 5, 9 and 15. Only cells with three or more signals from at least two of three investigated chromosomes were classified as hyperdiploid. ArrayCGH and qRT-PCR were focused on chosen list of mitotic genes, according to their role in normal centrosome duplication process.The frequency of MM cases positive for CA was 35% (35/100) and 39% (32/82), based on the analysis of PC samples and B-cell samples, respectively. Overall, 18% (9/50) of MM patients were double-positive. Presence of centrosome amplification in B-cells of MM patients was established in our previous study. Significant correlation of centrosome amplification in PCs with hyperdiploidy was not found. But association of CA in B-cells with PCs hyperdiploidy using phi 4-point correlation was proven (phi=0.358, p<0.05). In group of newly diagnosed patients (52/140), this correlation was much stronger (phi=0.555, p<0.05). ArrayCGH analysis of genes controlling centrosome duplication did not show correlation between their copy number defects and hyperdiploidy in myeloma cells. As for gene expression analysis, significant differences were found in levels of ARKA and PCNT genes (p<0.05). Relative quantification coefficient R of these genes was two times higher in CA positive patients when compared to CA negative patients. No significant correlation between amount of CA positive PCs and B-cells was found (p>0.05). But after splitting patients based on CA threshold, significant correlation was identified (r=-0,763, p=0.017) in double-positive group.In our study, we show association of CA in B-cells with PCs hyperdiploidy. This finding relates to the role of B-cell mitotic disruption in MM aneuploidy and cell carcinogenesis. It gives us a possibility to suspect the impact of abnormal B cells in myeloma cells development. B-cells with CA probably enter mitosis but do not finish it properly resulting in aneuploid cells; these cells may represent an aneuploid pool of MM cells.This study was supported by grants MSM 0021622434 and IGA 10207-3 from the Departments of Education and Health of the Czech Republic.No relevant conflicts of interest to declare.

Abstract 2952

Abstract 2968

The presence of chromosomal abnormalities detected by FISH in plasma cells is considered to be an important prognostic factor for patients with multiple myeloma. In this study we analysed the impact of gain(1)(q21), del(13)(q14), del(17)(p13), t(4;14) and (non)hyperdiploidy on prognosis in MM patients. Taking together 194 MM patients (median age 66 years) were successfully examined by FISH for presence of above named chromosomal abnormalities in plasma cells. A total of 47 patients (median follow-up 43.2 months; 91% first line treatment, 9% second line treatment) were treated by peripheral blood stem cells transplantation (PBSCT). A total of 86 patients (median follow-up 38.4 months; 38% first line treatment, 43% second line treatment, 19% >2 previous treatment lines) were treated by thalidomide based regimen (86% together with glucocorticoids and alkylating agens). A total of 61 patients (median follow-up 46.5 months; 16% first line treatment, 36% second line treatment, 33% third line treatment, 15% >3 previous treatment lines) were treated by bortezomib based regimen (53% with glucocorticoids and alkylating agens, 20% with glucocorticoids + anthracycline). Gain(1)(q21) was found in 52% (101/194), del(13)(q14) in 55% (69/125), del(17)(p13) in 12% (13/112), t(4;14) in 20% (18/90) and 44% (56/126) of cases were hyperdiploid. In any of the three treatment based groups of patients we haven't found any significant difference in TTP, OS and treatment response (ORR) between any studied positive/negative chromosomal abnormality except gain(1)(q21). See Table 1 for results overview (TTP only). In patients treated by PBSCT (regardless of their pre-treatment) significant difference in TTP and OS for positive/negative patients was observed (15.6 vs. 27.3 months, p=0.002 for TTP; 47.2 vs. NR months, p=0.001 for OS). Furthermore, we entirely focused on newly diagnosed patients divided them into three subgroups: (1) patients treated by PBSCT in first line (n=42), (2) patients treated by thalidomide based regimen in first line (n=33) and (3) patients treated by bortezomib based regimen in first line (n=10). We confirmed unfavourable impact of gain(1)(q21) in patients treated with PBSCT whereas TTP and OS in gain(1)(q21) positive/negative patients was 15.6 vs. 27.3 months, p=0.003 (TTP); 47.2 vs. 66.9 months, p=0.002 (OS). This difference in TTP/OS among gain(1)(q21) positive/negative patients was observed neither in thalidomide based subgroup [NR vs. 11.7 months, p=0.956 (TTP); NR vs. 34.4 months, p=0.683 (OS)] nor in bortezomib based subgroup [(8.5 vs. 6.5 months, p=0.549 (TTP); NR vs. 6.8, p=0.254 (OS)]. See Table 2 for results overview (TTP only). In this study we revealed gain(1)(q21) almost in one half of MM cases, and we defined the newly diagnosed MM patients carrying gain(1)(q21) as a unique group of patients with poor prognosis. Nevertheless, treatment based on bortezomib / thalidomide combination probably suppresses unfavourable prognostic impact of all studied chromosomal abnormalities including gain(1)(q21). This study was supported by grant LC06027 of Masaryk University, Brno, Czech Republic, and by grants MSM0021622415 and MSM0021622434 of Ministry of Education, Czech Republic, and by IGA grants NR/9317-3 and NS/10207-3/2009 of Ministry of Medicine, Czech Republic.No relevant conflicts of interest to declare.

Abstract 2820

Amplification of chromosome band 1q21 as well as increased expression of CKS1B gene in this area is a frequently mentioned prognostic factor for patients with multiple myeloma (MM). Total 39 newly diagnosed multiple myeloma patients (median age: 56 years) enrolled in Faculty Hospital Brno, Brno, Czech Republic, were examined for 1q21 amplification status. All patients received 4 cycles of vincristine, adriamycin and dexamethasone (VAD) as induction and one course melphalan 200mg/m2 followed by autologous stem cell transplantation (ASCT). The median follow-up from treatment start was 16.1 (range: 2.2–44.0) months. All the “end-point” intervals and treatment responses were assigned by IMWG criteria. Plasma cells were identified by cytoplasmic light-chain immunofluorescence followed by fluorescence in situ hybridisation (cIg-FISH). Amplification of 1q21 (Amp(1q21)) was assigned utilizing labelled BAC clone (RP11-205M9) DNA probe. Cut-off level for Amp(1q21) was established to 10% of total amount of cells with additional signals detected. Amp(1q21) was found in 41% (16/39) patients. Clinical parameters valid for patients with Amp(1q21) versus patients lacking Amp(1q21) were as follows: overall response rate (ORR) achieved 87.5% (14/16) vs. 91.3% (21/23) patients (p=0.404); overall survival (OS) median was 22.4 months vs. not yet reached (p=0.022); time to progression (TTP) median was 16.1 months vs. not yet reached (p=0.010); progression-free survival (PFS) median was 15.6 months vs. 25.2 months (p=0.023) and duration of response (DOR) median was 15.9 months vs. not yet reached (p=0.048). There were found statistical significant difference in all named “end-point” intervals (OS, TTP, PFS and DOR) between patients with/without Amp(1q21) but not in ORR. In conclusion, patients with Amp(1q21) treated by ASCT have significant shorter PFS median (15.6 months) when compared with patients lacking Amp(1q21) with PFS median 25.2 months (p=0.023). This findings is in accordance with previously published work (Chang et al., 2006).

Amplification of chromosome band 1q21 as well as increased expression of CKS1Bgene in this area is a frequently mentioned prognostic factor for patients with multiple myeloma (MM). Total 39 newly diagnosed multiple myeloma patients (median age: 56 years) enrolled in Faculty Hospital Brno, Brno, Czech Republic, were examined for 1q21 amplification status. All patients received 4 cycles of vincristine, adriamycin and dexamethasone (VAD) as induction and one course melphalan 200mg/m2followed by autologous stem cell transplantation (ASCT). The median follow-up from treatment start was 16.1 (range: 2.2–44.0) months. All the “end-point” intervals and treatment responses were assigned by IMWG criteria. Plasma cells were identified by cytoplasmic light-chain immunofluorescence followed by fluorescence in situhybridisation (cIg-FISH). Amplification of 1q21 (Amp(1q21)) was assigned utilizing labelled BAC clone (RP11-205M9) DNA probe. Cut-off level for Amp(1q21) was established to 10% of total amount of cells with additional signals detected. Amp(1q21) was found in 41% (16/39) patients. Clinical parameters valid for patients with Amp(1q21) versus patients lacking Amp(1q21) were as follows: overall response rate (ORR) achieved 87.5% (14/16) vs. 91.3% (21/23) patients (p=0.404); overall survival (OS) median was 22.4 months vs. not yet reached (p=0.022); time to progression (TTP) median was 16.1 months vs. not yet reached (p=0.010); progression-free survival (PFS) median was 15.6 months vs. 25.2 months (p=0.023) and duration of response (DOR) median was 15.9 months vs. not yet reached (p=0.048). There were found statistical significant difference in all named “end-point” intervals (OS, TTP, PFS and DOR) between patients with/without Amp(1q21) but not in ORR. In conclusion, patients with Amp(1q21) treated by ASCT have significant shorter PFS median (15.6 months) when compared with patients lacking Amp(1q21) with PFS median 25.2 months (p=0.023). This findings is in accordance with previously published work (Chang et al., 2006).