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Chronic lymphocytic leukemia (CLL) is the most common blood cancer in adults and known for its heterogeneous disease course. The nature of CLL calls for new studies of pathogenetic variables validated by prognostic impact. Here we propose a new biological classification system of CLL that associates normal B cell subsets gene expression (GE) signatures (BAGS) with GE in CLL. We used fluorescence activated cell sorting, GE profiling, and statistical modeling to generate a B-cell associated gene signature (BAGS) classifier for pre-BI, pre-BII, immature, naive, memory, and plasma cells harvested from healthy bone marrow samples during cardiac surgery. We used the Gene Expression Omnibus (GEO) to identify 8 independent CLL cohorts with available microarray data generated on compatible array platforms. Altogether, the study included 1239 patients, who were untreated at sampling for microarray analysis. The classifier was established using regularized multinomial regression on median-centered GE profiles from the normal B cell subsets and consisted of 184 genes with 27-54 genes per subtype. Each patient was assigned to a BAGS subtype (pre-BI, pre-BII, immature, naive, memory, and plasmacell) according to the highest probability score of the classifier. The minimum probability threshold for confident classification was chosen such that 15% remained unclassified. BAGS subtype frequencies in the CLL cohorts were analyzed using Fisher's exact test and revealed a distribution pattern that did not vary significantly between the 8 cohorts (P = 0.22) (Fig 1). Most samples were classified as memory (28.1-45.5%), followed by naive (15.2-32.3%), pre-BI (10.0-17.6 %), immature (0.0-8.7%), plasmacell (1.8-4.6%), and pre-BII (0.0-3.9%). To validate the prognostic impact, our primary endpoint was time to treatment start and was evaluated using the Kaplan Meier method using available data (n = 68). We saw a significant shorter time to treatment start amongst patient with a pre-germinal center CLL subtype (pre-BI, pre-BII, immature, and naive) compared to post-germinal center subtypes (memory and plasmacell) (P = 0.03) (Fig 2). Next, BAGS subtype was associated with the IgVH mutational status (n = 222), which showed that a larger proportion of post-germinal center subtypes had mutated IgVH genes (73.4%), whereas pre-germinal center subtypes were just as likely to have unmutated IgVH genes (48.8%) as mutated IgVH genes (52.2%). Finally, to investigate biological differences between the BAGS subtypes, we identified differentially expressed genes (DEG) in each subtype versus the rest. Fisher's method was used to combine p-values for statistically significant DEGs in a metaanalyis including all cohorts. Of known therapeutic CLL targets, CD20 and CD52 were both significantly upregulated in the memory BAGS subtype compared to the rest. Using a phenotypic cell of origin approach, our findings show that CLL samples display end-stage phenotypes resembling the whole spectrum of normal B-cells and indicate transcriptional plasticity as a trait that may contribute to the heterogeneity observed in CLL. Figure 1 Figure 1. BAGS assignment of CLL samples.The distributions and frequencies of assigned BAGS subtypes are shown for all 8 CLL cohorts. Colors: pre-BI (purple), pre-BII (light green), immature (green), naive (turquoise), memory (orange), plasma cells (blue), unclassified (grey). Tests for significantly different distributions across data sets were calculated using Fisher's exact test (P = 0.22). Figure 2 Figure 2. Time to first treatment for pre- and postgerminal BAGS subtypes. Kaplan Meier plot showing years from gene expression profiling until first treatment for the pre- and post-germinal center BAGS subtypes. Unclassified samples are not included. The Munich cohort (n = 68) was used. P = 0.03. Disclosures El-Galaly: Roche: Consultancy, Other: travel funding.

Recently, high-dose chemo- and radiotherapy for newly diagnosed young patients with multiple myeloma has been established in member centers of the Nordic Myeloma Study Group (NMSG). The tr...

The aim of this study was to evaluate the efficacy, safety and toxicity of short-term priming with recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately after diagnosis but before combination chemotherapy (CHOP) for non-Hodgkin's lymphomas. Of fourteen patients entering the study, seven received five days subcutaneous injection of rhG-CSF (5 micrograms/kg/day) before CHOP (CSF-group), and seven were treated with CHOP alone (control group). Blood samples were studied before and on days 1-5 during rhG-CSF priming as well as twice weekly after treatment. The number of blood and bone marrow progenitors was identified by clonogenic growth day 7, 14 and 21 of GM-CFU in semisolid medium. Blood absolute neutrophil counts increased in all rhG-CSF primed patients. The expansion of marrow myelopoiesis resulted in increased myeloid:erythroid ratios, increased bone marrow cellularity and increased numbers of myeloid progenitors both in the blood as well as the marrow. Chemotherapy induced neutropenia developed on day 9-12 in all patients independent of myeloid growth factor priming. However, neutropenia appeared earlier in the cytokine primed group (P = .0038). Five patients in the CSF-group and three patients in the control group were hospitalized with neutropenic fever, and septicemia was documented in three patients in the CSF-group. RhG-CSF induced expansion of myelopoiesis immediately before combination chemotherapy mobilized sufficient number of blood progenitors for apheresis but did not result in reduction of duration and degree of neutropenia in patients with newly diagnosed non-Hodgkin's lymphoma. Although the small number of patients prevents drawing definite conclusions, this time schedule for priming should be used with caution in the future due to an increased risk of hematologic toxicity.

Background Malignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq. Results We identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes - CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection. Conclusion A sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.

Background: Individual cellular heterogeneity within the acute myeloid leukemia (AML) bone marrow samples can be observed by multi parametric flow cytometry analysis (MFC) indicating that immunophenotypic screening for leukemic blast subsets may have prognostic impact. Material and methods: Samples from de novo AML patients of all cytogenetic risk groups were collected at diagnosis and subjected to MFC based on a four-color antibody panels against 33 CD membrane markers and retrospectively analyzed for the leukemia blast expression pattern and mean fluorescence intensity. Identification of the leukemic blast cells was based on right angle light scatter (SSC) and expression of CD45 and the cellular heterogeneity identified by the presence of at least two distinct subsets by any CD marker. Results: Analysis of marrow samples from 86 patients with cytogenetic intermediate risk identified recurrent heterogeneous blast phenotypes for selected CD markers, three of which had prognostic impact with loss or gain of CD58, CD117, or CD14 expression. Multivariate Cox regression analysis of diagnostic variables identified poor prognostic factors: Age >55 years, presence of extramedullary disease, WHO performance score >2, a heterogeneous CD58, CD117, or CD14 expression on blast cells. Each variable added to a simple and clinical useful and MFC based prognostic score system associated to inferior survival in the intermediate risk group of AML patients. Conclusions: These observations support that leukemic blast heterogeneity detected by MFC has additional prognostic significance in de novo AML; however, the score system needs to be prospectively validated in future clinical trials before implementation. © 2012 International Clinical Cytometry Society

The cancer stem cell (CSC) concept has important therapeutic implications, but its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined. Evidence of their heterogeneous origins, frequencies and their genomic, as well as their phenotypic and functional, properties has added to the confusion and has fuelled new ideas and controversies. Participants in The Year 2011 Working Conference on CSCs met to review these issues and to propose a conceptual and practical framework for CSC terminology. More precise reporting of the parameters that are used to identify CSCs and to attribute responses to them is also recommended as key to accelerating an understanding of their biology and developing more effective methods for their eradication in patients.

While the etiology of multiple myeloma (MM) is largely unknown, evidence for an inherited genetic susceptibility is provided by the two-fold increased risk of the disease seen in first-degree relatives of cases of MM. It is likely that part of this heritable risk is a consequence of the co-inheritance of low-risk genetic variants. The accumulated experience to date in identifying risk variants for other tumors has highlighted difficulties in conducting statistically and methodologically rigorous studies. The MyelomA Genetics International Consortium (MAGIC) includes 16 research groups in Europe, Asia, Australasia, the Middle East and the Americas engaged in studying the genetics of MM. The first goal of MAGIC is to identify and characterize common genetic variants for MM through association-based analyses. Here, we review the rationale for identifying genetic risk variants for MM and our proposed strategy for establishing MAGIC.

Abstract 2637 Background Hodgkin lymphoma (HL) has traditionally been regarded as a disease primarily affecting the lymphatic organs. The introduction of dual modality [18]F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) may challenge this statement as PET/CT reveals more sites of extranodal disease than CT alone. The aim of this study was to characterize the clinico-pathological and prognostic features of newly diagnosed HL patients with PET/CT-ascertained skeletal involvement. Patients and Methods We performed a retrospective analysis of the pre-therapeutic PET/CT results from newly diagnosed HL patients at four Danish university hospitals. Criteria for inclusion in the study were (a) histologically verified HL, (b) age ≥15 years, and (c) PET/CT-based staging. Clinical information was obtained from the Danish Lymphoma Registry (LYFO) supplemented by review of medical records. Imaging reports were reviewed for extent of disease. Patients were divided into three groups according to their pattern of PET/CT-positivity: HL confined to lymphatic organs (lymph nodes, spleen, thymus, Waldeyer's ring) (group 1); HL with focal skeletal PET/CT lesions as the only extranodal involvement (group 2); HL with any extranodal involvement, including skeletal lesions if other extranodal sites were concurrently involved (group 3). Results A total of 454 patients with newly diagnosed HL were included and the median follow-up time was 34 months (range 1–113). Focal skeletal PET/CT lesions were identified in 82 patients (18%). HL was confined to nodal areas in 336 (74%) patients (group 1). Among the 118 (26%) patients with extranodal disease, 39 had focal skeletal PET/CT lesions as their only extranodal manifestation (group 2), while 79 had involvement of other extranodal sites with or without co-existing skeletal lesions (groups 3). Group 1 patients exhibited the most favorable combination of clinico-pathological features, group 3 patients displayed most adverse combination of clinico-pathological features, whereas group 2 was intermediate (Table). In a 1:1 comparison between group 1 and 2 patients, the latter had significantly lower hemoglobin (Hgb) levels, higher erythrocyte sedimentation rates (ESR) and higher occurrence of B-symptoms. The PFS for group 2 patients was similar to that of group 3 patients (p=0.85), but significantly shorter than that of group 1 patients (p Conclusions PET/CT-based identification of focal skeletal lesions as the only extranodal involvement in patients with newly diagnosed HL is not uncommon. This group of patients frequently presents with systemic symptoms and elevated ESR. In addition, their PFS is inferior to that of patients with nodal HL, but similar to that of HL patients with other types of extranodal disease. Although rarely histologically verified, these focal skeletal lesions are thus likely to represent extranodal manifestations of HL. Disclosures: Hutchings: Takeda/Millennium: Consultancy.

Purpose To investigate whether bone marrow biopsy (BMB) adds useful information to [18F]fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) staging in patients with Hodgkin lymphoma (HL). Patients and Methods Newly diagnosed patients with HL undergoing a pretherapeutic staging that encompasses both PET/CT and BMB were included in this retrospective study. The pattern of skeletal FDG uptake was categorized as uni-, bi-, or multifocal (≥ three lesions). Clinical stage, risk assessment, and treatment plan were determined with and without the contribution of BMB results according to the Ann Arbor classification and the guidelines from the German Hodgkin Study Group. Results A total of 454 patients with HL were included of whom 82 (18%) had focal skeletal PET/CT lesions and 27 (6%) had positive BMB. No patients with positive BMB were assessed as having stage I to II disease by PET/CT staging. BMB upstaged five patients, assessed as being stage III before BMB; none of the 454 patients would have been allocated to another treatment on the basis of BMB results. Focal skeletal PET/CT lesions identified positive and negative BMBs with a sensitivity and specificity of 85% and 86%, respectively. The positive and negative predictive values of focal skeletal PET/CT lesions for BMB results were 28% and 99%, respectively. Conclusion A consistent finding of this study was the absence of positive BMBs in PET/CT-assessed stage I to II disease. The omission of staging BMB would not have changed the risk assessment or treatment strategy in this cohort of 454 newly diagnosed patients with HL.

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.

The t(4:14) translocation affects two potential oncogenes, FGFR3 and MMSET, in multiple myeloma (MM). We investigated the frequency of FGFR3 dysregulation and its prognostic value in MM. FGFR3 mRNA levels were determined in 110 diagnostic bone marrow (BM) samples from MM patients. In addition, selected BM samples were screened for elevated MMSET mRNA levels. 14.5% (16/110) of MM BM samples showed dysregulated FGFR3 expression. Follow-up of 76 MM patients showed no significant difference between FGFR3 dysfunction and survival (P = 0.3) or correlation with known prognostic factors. Further, no linear relation was observed between FGFR3 and MMSET levels.

Peripheral blood-localized clonal cells in patients with multiple myeloma (MM) have been reported to be insensitive to chemotherapy and it has been suggested that these cells may include the proliferative compartment in myeloma. In this study, circulating clonal CD19 + cells levels were determined in 10 patients with MM by performing patient-specific RT-PCR on single flow-sorted CD19 + cells. A variable proportion of CD + cells being clonal were identified (0.101-6.130%, mean 1.019%). In 10/10 MM patients clonal CD19 + cells were found after high-dose treatment and peripheral blood stem cell transplantation. In conclusion, CD19 + clonal cells include a population of cells persistent after high dose chemotherapy that may be responsible for relapse.

Post-therapy surveillance imaging in patients with lymphoma remains controversial. We report our experience with positron emission tomography/computed tomography (PET/CT) surveillance in patients with aggressive non-Hodgkin lymphoma in first complete remission (CR). The 138 PET/CTs performed in 52 patients revealed four unsuspected relapses. In one patient, relapse was visualized by fluorodeoxyglucose (FDG) accumulation without any significant CT pathology. The specificity and sensitivity of surveillance PET/CT were 89% and 100%, respectively. The predictive values of positive and negative PET/CTs were 21% and 100%, respectively. The cost of half-yearly routine PET/CT surveillance during the first 2 years in CR was $US8552 per patient and accounted for 81% of the total follow-up costs. PET/CT was effective in detecting unexpected relapse and normal PET/CT supported continuous CR. However, the impact of PET/CT was limited by the high number of false-positive results and PET/CT surveillance was costly compared to CT surveillance.

Background: Fracture healing includes formation of cartilage, blood vessels and bone, which involves circulating progenitor cells, cytokines and growth factors in a complex homeostasis of tissue regeneration. Here we describe a clinical study of circulating cellular and humoral variables by a time dependent multiparametric approach following traumatic lesions. Materials and Methods: Two prospective cohorts of 50 patients, with ankle- or hip fracture (cohort 1) or planned hip replacements (cohort 2) were studied as was age matched healthy controls. Blood samples were timely collected during the post traumatic period and analysed for i) non-haematopoietic (CD45neg) mesenchymal subsets by multiparametric flow cytometry (MFC), ii) global gene expression profiling (GEP) by micro array and iii) serum inflammatory markers including the growth factor YKL-40 by immunoassays (ELISA). Integrated analyses were performed to identify cellular and humoral patterns with potential impact on tissue regeneration. Results: Posttraumatic levels of circulating white blood cells, immature progenitor subsets and platelets as well as YKL-40, IL-6 and CRP varied biphasic and correlated to type of traumas. Analytic MFC identified two minor CD45neg blood compartments which simultaneously expressed varying degrees of CD105, CD133, CD73, VEGF-R, CD144, or CD31, CD34, CD166, CXCR4, respectively, supporting that mesenchymal subsets are involved in fracture healing. Analysis by microarray identified posttraumatic significant changes in gene expression of specific genes with known relation to inflammation, bone regeneration and angiogenesis in circulating mononuclear cells (MNC). ELISA quantitation of YKL-40 revealed posttraumatic changes higher in hip traumas compared to patients with ankle fractures (MNC: p=0.0006; YKL-40: p=0.0004). YKL-40 also correlated to the type of bone trauma, documented by different levels in patients with planned surgical hip replacements and traumatic hip fractures (p=0.005). Conclusions: The present study describes a posttraumatic time dependent cellular and humoral response after planned hip replacements, ankle and hip fractures. The data identify and enumerate potential mesenchymal progenitor cells supporting a regenerative role. Finally the analysis documented a correlation between the growth factor YKL-40 and bone traumas separating it from IL-6 and CRP. These observations can be used to future identification, isolation and characterization of circulating cells with impact in bone regeneration.

Abstract 2627 Accurate staging is essential for the optimal choice of first-line treatment in Hodgkin lymphoma (HL). Bone marrow biopsy (BMB) is still widely recommended as part of the routine staging workup. However, this procedure may not be necessary for patients staged with [18]F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT). The aims of this study were; 1) to determine if bone marrow infiltration can be excluded based on staging PET/CT result, and 2) to investigate whether BMB still adds useful information in the PET/CT era. Patients with newly diagnosed HL undergoing pre-therapeutic staging at three Danish university centers were eligible for inclusion in this retrospective study, provided both PET/CT and BMB were performed at staging. Patients were identified in the Danish Lymphoma Registry (LYFO). BMB results were first obtained from LYFO and validated by a review of local pathology records. PET/CT records were retrieved and reviewed. The patterns of skeletal FDG uptake were categorized as follows: 1) uni-focal, 2) bi-focal, 3) multi-focal or 4) diffuse homogeneous FDG uptake. Clinical stage and risk assessment were performed with and without the account of BMB results, according to the Ann Arbor classification, the International Prognostic Score (IPS), and the German Hodgkin Study Group risk criteria for limited disease. A total of 392 HL patients (Aalborg = 59, Aarhus = 67, Rigshospitalet = 266) were included, of whom 372 patients had classical HL and 20 had nodular lymphocyte predominant HL. According to staging PET/CT, 203 patients had limited stage disease and 189 patients had advanced stage disease. The median age at diagnosis was 39 years (range 15–87) and the female:male ratio was 3/4. BMB was positive for HL in 24 patients (6%), all of whom had been independently assessed by PET/CT as stage III (n=5) or IV (n=19) disease. Thus, no patients with PET/CT stage I-II disease, irrespective of the presence of B-symptoms, had HL infiltration in the BMB (p Skeletal FDG accumulation was seen in 90 patients and characterized as uni-focal (n=15), bi-focal (n=8), multi-focal (n=45) or diffuse (n=22). Among the 68 patients with focal skeletal FDG accumulation, 20 patients had positive BMB as compared to none of the patients with diffuse FDG uptake. Sixty out of 68 patients with focal skeletal FDG uptake had PET/CT response assessment during therapy. All patients who responded to therapy showed resolution of the pathological skeletal FDG uptake. The presence of focal skeletal FDG accumulation identified HL infiltration in the BMB with a sensitivity and specificity of 83% and 87%, respectively. The positive and negative predictive values of focal skeletal FDG accumulation for HL infiltration in the BMB were 29% and 99%, respectively. With both positive BMB and focal skeletal FDG accumulation considered to reflect true bone/bone marrow involvement, the sensitivity for bone/bone marrow disease was 94% for PET/CT and 33% for BMB. In terms of prognostic information, BMB upstaged 1% of the patients (n=5) from stage III to stage IV, with a one-point change of the IPS as an additional consequence. However, none of the 392 patients were allocated to another risk group or treatment group on the basis of BMB results. At present there is no optimal gold standard for diagnosing bone/bone marrow disease in HL. Unilateral iliac crest BMB examines a very limited part of the total bone marrow volume, while PET/CT visualizes abnormal FDG accumulation throughout large parts of the skeletal system. Whether abnormal skeletal FDG uptake represents HL in all cases remains to be clarified. The most consistent finding of the present study was the absence of positive BMBs in PET/CT assessed stage I-II disease, where routine BMB seems to have no relevance. Furthermore, the omission of staging BMB would have had no impact on the treatment strategy in this cohort of 392 newly diagnosed HL patients. Disclosures: No relevant conflicts of interest to declare.

CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) ± rituximab [(± R)CHOP] is the current standard of care for aggressive non-Hodgkin lymphoma (NHL). Anemia resulting from chemotherapy can be treated with erythropoiesis-stimulating agents (ESAs). As part of the observational IMPACT NHL study, data were collected on ESA use and anemia-related outcomes in 1829 adults receiving (± R)CHOP-14 or (± R)CHOP-21. Overall, 33% of patients were anemic during chemotherapy. Older age, lower baseline hemoglobin (Hb), worse performance status, more advanced disease stage, and use of CHOP-14 were significant predictors of transfusion and anemia in logistic regression models. ESAs were received by 404 patients, usually in response to low or declining Hb levels. Most patients (65%) had Hb 9-11 g/dL at ESA initiation, and 89% (Kaplan-Meier percentage) achieved Hb 10-12 g/dL. In conclusion, two-thirds of anemic patients with NHL receiving (± R)CHOP initiated ESA treatment at Hb 9-11 g/dL, and most achieved target Hb levels (10-12 g/dL).

Abstract SCI-5 Multiple myeloma (MM) is a malignant disorder characterized by the (mono)clonal expansion of terminally-differentiated plasma cells (M-PC) in the bone marrow (BM) that produce and secrete a monoclonal immunoglobulin (Ig), detectable in the serum and/or urine. Infiltration by the expanded M-PC is easily identified in the involved tissues, through conventional morphology and immunophenotyping. However, the possibility exists that rather than M-PC, a less differentiated B-cell that represents a minor fraction of all tumor cells and retains self-renewal properties, is responsible for the outgrowth of the more differentiated M-PC compartment. In B-cell disorders, the idiotypic Ig produced by tumor cells and defined by its CDR3 sequence, acts as a genetic fingerprint for clonally-related B-cells. In MM, the specificity of the idiotype is further enhanced through modification of V genes by somatic hypermutation (SHM) at the germinal center (GC), since M-PC display extensively mutated VH genes which are stable throughout the disease. This suggests that in MM, malignant transformation could occur in a post-GC B-cell. Based on the CDR3 sequences of the Ig genes of M-PC, preliminary studies have identified tumor-associated circulating peripheral blood (PB) CD19+ B-cells, whose malignant/clonogenic potential remained to be demonstrated. More recently, the Matsui group has reported that while in MM cell lines both CD138− and CD138+ cells retain clonogenic capacity after in vitro serial plating clonogenic assays, in primary MM samples, such (ex vivo and in vivo) ability would be restricted to the CD34−/CD138− compartment. Of note, the clonogenic growth of these later cells significantly decreased after depletion by CD19, CD22, CD20 and CD45 antibodies, and it was associated with an in vitro multidrug-resistant functional phenotype (restricted to CD19+/CD27+, CD138− cells but not CD138+ cells) and the Hedgehog (Hh) stem cell-associated signaling pathway. These results point out the potential existence of a CD19+, CD20+, CD138− pre-PC compartment responsible for the expansion of M-PC in MM. In turn, evidence also exists in both the SCID-Hu model and in Rituximab treated MM patients, which suggests that plasmablasts/PC -but not pre-plasmablasts-, could act as MM “stem” cells, the precise characteristics of such cells remaining to be precisely defined. Alternatively, it could also be possible that both cell cellular components coexist and are relevant to MM progression through appropriate interaction with the BM stroma. Independently of all the above, trafficking of such cells through PB to BM niches could also play a key role in the spread of the tumor and its malignant behavior. In this regard, we recently confirmed that a relatively high percentage of MM patients (and a substantial fraction of all MGUS cases) show circulating PB PC with i) tumor-related clonal VH gene rearrangements and ii) an aberrant immunophenotypic profile which largely overlaps with that of BM M-PC from the same subjects; the only minor differences consisted of a significantly lower expression of CD38 and CD138, smaller size and internal complexity, features that indicate a slightly more immature plasmablastic/PC profile. Noteworthy, this was the only PB B-cell compartment for which clonally-related B-cells were detected with a sensitivity of Disclosures: Sonneveld: Millennium: Consultancy; Celgene: Consultancy. Orfao:Becton/Dickinson Biosciences Europe: Patents & Royalties, Research Funding; Cytognos SL: Patents & Royalties; Alexion: Membership on an entity's Board of Directors or advisory committees; Vivia Biotech: Research Funding; Mundipharma: Research Funding.

Udgivelsesdato: 2010-Aug-1 BACKGROUND: An unexplained survival difference was observed in the Nordic Myeloma Study Group (NMSG) high-dose therapy trial 5/94 in Denmark compared with Sweden and Norway; however, this difference was eliminated in the subsequent NMSG trial 7/98. It was hypothesized that a detailed analysis of potential explanations would reveal important information for future designs of clinical trials for multiple myeloma (MM) patients in Denmark. PATIENTS AND METHODS: The analysis is based on 3 consecutive clinical trials coordinated by NMSG from 1990 to 2000: NMSG 4/90 including 583 patients, NMSG 5/94 including 274 patients and NMSG 7/98 including 414 patients with newly diagnosed MM. Event-free and total survival rates were calculated according to the Kaplan-Meier method, and survival comparisons were made by the log-rank test. The Cox proportional hazards regression model was used to estimate the prognostic importance of selected variables. RESULTS: The analysis revealed no differences in disease stages, prognostic variables, or inclusion bias at diagnosis between the 3 consecutive NMSG trials. However, the number of initial treatment failures was low, and post-relapse survival was superior in Swedish patients as compared to Danish patients. These differences were explained by a defensive clinical practice in Denmark during 1994-1997 for patients with poor risk refractory or relapsed disease. CONCLUSION: These initially observed differences were subsequently eliminated most likely as a consequence of international collaboration improving diagnosis, research infrastructure, clinical training, and education as planned within the European Myeloma Network (EMN).

BackgroundRecent reports indicate that in vitro drug screens combined with gene expression profiles (GEP) of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM) a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information.Materials and methodsMicroarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA), sparse partial least squares (SPLS) and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis.Principal findingsBoth cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value.ConclusionThe present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to better understand the mechanism underlying the preparedness to melphalan resistance.

The introduction of the Immunomodulatory drugs (IMiDs) and proteasome inhibitors, used either as a single-agent or combined with classic anti-myeloma therapies, has improved the outcome for patients with relapsed myeloma. However, there is currently no generally accepted standard treatment for relapsed/refractory myeloma patients, partly because of the absence of trials comparing the efficacy of the novel agents in relapsed/refractory myeloma. Choice of a new treatment regimen depends on both patient and disease-specific characteristics. A lenalidomide-based regimen is the first choice in patients with neuropathy, while bortezomib has the highest efficacy in patients with renal insufficiency and is not associated with increased risk of thromboembolism. A second autologous stem cell transplantation (auto-SCT) can be applied in patients with a progression-free period of >= 18-24 months after the first auto-SCT. In high-risk relapse such as occurring early after auto-SCT consolidation with allogeneic SCT can be considered. In this review we provide an overview of the various salvage regimens and give recommendations for treatment of patients with relapsed/refractory myeloma in different clinical settings. (C) 2010 Elsevier Ltd. All rights reserved.

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) na?̈ve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL). © 2010 International Clinical Cytometry Society., Grant sponsor: MSCNet European strep; Grant number: N_ E06005FF; Grant sponsor: Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain; Grant numbers: RTICC RD06/0020/0035, PI06/0824, PS09/02430; Grant sponsor: Gerencia Regional de Salud de castilla y León; Grant number: GRS206/A/08; Grant sponsor: Ayuda GR37 de Excelencia de Castilla y León, Consejeria de Educación, Junta de Castilla y León, Valladolid, Spain; Fundación Samuél Solorzano, Salamanca, Spain; Grant number: FS/16-2008.

Over the past two decades, not only treatment options, but also the diagnosis, staging, and risk assessment of multiple myeloma (MM), have undergone significant development, partially due to a deeper understanding of MM pathogenesis. Conventional cytogenetics and fluorescence in situ hybridization are routinely assessed in MM, and when combined with ISS stage may attain an even better predictive potential. In order to achieve even more effective and individualized therapies, one crucial goal is the identification of genes and gene combinations that predict for response or resistance to chemotherapy. High-dose chemotherapy with autologous stem cell transplant (SCT) still remains the standard therapy for younger patients, with novel agents now being included in both pre-transplant regimens and post-transplant consolidation/maintenance approaches. Similarly, novel agents are also being incorporated into allogeneic SCT for selected patients. In the treatment of elderly patients with MM, novel agents have been successfully incorporated into less intensive regimens, including melphalan/prednisone, low-dose dexamethasone, and cyclophosphamide/dexamethasone. While second-generation proteasome inhibitors are currently being intensively investigated, the subcutaneous administration of bortezomib, being equivalent to the established i.v. route, is now entering clinical practice. Supportive care remains a crucial aspect in the management of MM. The European Myeloma Network Trialist Group aims to address these contemporary aspects in MM.

Recent studies concerning the numbers of circulating clonal B cells in patients with multiple myeloma (MM) have reported conflicting data regarding the exact level and phenotype of clonal B cells and their response to treatment. In this report we document that the peripheral blood tumor burden at presentation was reduced by induction therapy to a low level, regardless of the initial tumor burden. However, the residual clonal compartment persisted before and after transplant. The level of clonal cells showed no correlation with CD19+cell levels. In a single patient with MM, high numbers of phenotypically aberrant clonal cells with altered CD19 expression were identified.

Udgivelsesdato: 2009 Malignancies in the haematopoietic system seem to depend on a small subset of so-called cancer stem cells (CSC) for their continued growth and progression - this was first described as the "sleeper-feeder theory" for leukaemia. The leukaemia stem cell was the first of such subsets to be described although the origins of these cells have been difficult to dissect. Consequently, their biology is not fully elucidated, which also holds true for the normal-tissue counterparts. The stem cell concept describes stem cells to be of low frequency, self renewing and with multilineage potential based on phenomenology - a definition which may not hold strictly true for CSCs when studied in animals and humans in vivo and in vitro. Several studies have analysed the cellular hierarchy of the haematopoietic system by cell sorting of few and even single cells, tracking acquired genetic changes and performing transplantation model studies to document subsets within the differentiating hierarchy as potential CSC compartments. In leukaemia the CSC has been described in the bone marrow compartment of haematopoietic stem cells (HSC); however, in other bone marrow disorders like multiple myeloma it is likely that the cell of origin is a more differentiated cell, like post-germinal memory B cells or plasmablasts. Studies performed so far have even indicated that the genetic events may occur in different B cell subsets in accordance with the stepwise oncogenesis of the disease. Although our understanding of the nature and biology of these initiating cells remains unknown, the obvious existence of such cells has implications for understanding initial malignant transformation and disease metastasis or progression and, most important, the selection of individualised therapeutic strategies targeting the subsets harbouring the CSC function. In the present review on stem cells in haematological malignancies we have focused on two topics, first, describing the stem cell concept in health and disease, and its "phenomenology", and second, describing the CSC compartments in leukaemia and multiple myeloma.

Udgivelsesdato: 2009 The impact of chemotherapy resistant B cells in multiple myeloma (MM) needs to be evaluated by in vivo targeted therapy. Here we report the conclusions from a phase II randomized, placebo controlled trial adding fludarabine to the induction with cyclophosphamide-dexamethasone. Based on an interim toxicity and safety analysis, the trial was stopped following inclusion of 34 of a planned 80 patients due to a reduced number of patients (4/17) actually harvested in the experimental arm compared to the control arm (11/17; p lower than 0.05). In conclusion, the scheduled fludarabine dosage in 2 cycles combined with alkylating therapy impairs stem cell mobilization and standard therapy in young MM patients and should not be administrated up-front.

Autologous stem cell transplantation is still considered the standard of care in young patients with multiple myeloma (MM). This disease is the most common indication for high-dose therapy (HDT) supported by hematopoietic stem cell transplantation and much data support the benefit of this procedure. Results of randomized studies are in favor of tandem autologous transplantation although the effect on overall survival is unclear. Based on sequential registration trials in the Nordic area, we aimed to evaluate the outcome of conventional single or double HDT. During 1994–2000 we registered a total of 484 previously untreated patients under the age of 60 years at diagnosis who on a regional basis initially were treated with single [Trial NMSG #5/94 and #7/98 (N = 383)] or double [Trial Huddinge Karolinska Turku Herlev (N = 101)] high-dose melphalan (200 mg/m2) therapy supported by autologous stem cell transplantation. A complete or very good partial response was achieved by 40% of patients in the single transplant group and 60% of patients in the double transplant group (p = 0.0006). The probability of surviving progression free for five years after the diagnosis was 25% (95% CL 18–32%) in the singletransplant group and 46% (95% CL 33–55%) in the double transplant group (p = 0.0014). The estimated overall five-year survival rate was 60% in the single transplant group and 64% in the double transplant (p = 0.9). In a multivariate analysis of variables, including single versus double transplantation, β2 microglobulin level, age, sex and disease stage, only β2 microglobulin level was predictive for overall survival (p > 0.0001) and progression free survival (p = 0.001). In accordance with these results, a 1:1 case-control matched comparison between double and single transplantation did not identify significant differences in overall and progression free survival. In this retrospective analysis up front double transplantation with melphalan (200 mg/m2) as compared to single transplantation did not seem to improve the final outcome among patients in the Nordic area. These data are in accordance with recent publications from the Bologna 96 trial indicating that a second transplant should not be recommended up front as standard care.

Recent studies concerning the numbers of circulating clonal B cells in patients with multiple myeloma (MM) have reported conflicting data regarding the exact levels of clonal B cells and the existence of clonal cells in the CD34 compartment. In this report we show that high numbers of clonal cells with a phenotype of late-stage B cells or pre-plasma cells were present in the peripheral blood (PB) of a patient with MM. During treatment the initial high level of PB clonal cells was markedly reduced and remained low (

A 40 year-old man with hairy cell leukemia (HCL) was treated with Cladribin, but achieved only partial response (PR). As expected, both 2 and 5 years later the disease had progressed and the patient was therefore twice retreated with Cladribin. A bone marrow examination after the last Cladribin course showed complete response with residual disease (CR-RD). The patient received consolidation therapy with Rituximab, achieved complete response (CR) and was still in CR 9 months later. Rituximab is a good treatment option for patients with refractory HCL because of its significant activity and minimal toxicity.

Udgivelsesdato: 2008-Mar BACKGROUND: Growth factors are frequently used to aid peripheral blood progenitor cell mobilization from bone marrow. This phase 2 study examined the efficacy and safety of pegfilgrastim for mobilizing peripheral blood progenitors cells for autologous transplantation. DESIGN AND METHODS: Patients with non-Hodgkin's lymphoma received one cycle of mobilizing chemotherapy (ifosfamide, carboplatin and etoposide, ICE). Twenty-four hours later they were randomized, double-blind, to receive a single dose of pegfilgrastim 6 mg or 12 mg, or filgrastim 5 mug/kg/day (until the end of leukapheresis). Following leukapheresis (collection phase), patients rested or received one or two 'salvage' cycles of ICE. High-dose BEAM chemotherapy was then given before peripheral blood progenitor cell transplantation. The primary end-point was the patients' mean yield of CD34(+) cells/kg during the collection phase. RESULTS: Ninety patients were randomized and received a study drug; 63% completed the collection phase. The patients' mean (95% CI) CD34(+) cell harvest per leukapheresis was 0.8 (0.5-1.4), 0.8 (0.5-1.6) and 1.2 (0.7-2.0)x10(6) cells/kg for the pegfilgrastim 6 mg, pegfilgrastim 12 mg and filgrastim groups, respectively. Twenty (69%), 17 (59%) and 23 (72%) patients in these three groups achieved the targeted minimum harvest (>/=2 x 10(6) cells/kg). The mean total harvests were 1.7, 1.4 and 2.2 x 10(6) cells/kg, respectively. Post-transplantation, the median days to absolute neutrophil count recovery (>/=0.5 x 10(9)/L) were 12, 11, and 11, respectively. Pegfilgrastim and filgrastim were generally well tolerated. CONCLUSIONS: Pegfilgrastim (6 or 12 mg) was effective for mobilizing peripheral blood progenitors cells in patients with non-Hodgkin's lymphoma. These data may aid the design of studies to clarify optimal dosing and leukapheresis with pegfilgrastim.

A 40 year-old man with hairy cell leukemia (HCL) was treated with Cladribin, but achieved only partial response (PR). As expected, both 2 and 5 years later the disease had progressed and the patient was therefore twice retreated with Cladribin. A bone marrow examination after the last Cladribin course showed complete response with residual disease (CR-RD). The patient received consolidation therapy with Rituximab, achieved complete response (CR) and was still in CR 9 months later. Rituximab is a good treatment option for patients with refractory HCL because of its significant activity and minimal toxicity. Udgivelsesdato: 2008

Udgivelsesdato: 2008-Jan-1 BACKGROUND: Today, intensive therapy that includes high-dose melphalan with autologous stem cell transplantation (ASCT) is considered standard therapy in younger patients with newly diagnosed myeloma. When the current trial was initiated, combined vincristine, doxorubicin, and dexamethasone (VAD) was the most commonly used induction therapy before ASCT and yielded rapid major responses without interfering with stem cell harvest. However, the administration of VAD demands a central venous access, and well-described toxicities are associated with the therapy. This randomized trial, which was initiated in 2001 by the Nordic Myeloma Study Group, was an attempt to bring a larger portion of patients to ASCT more quickly. METHODS: Patients were randomized to receive either 3 cycles of VAD or 2 courses of cyclophosphamide plus dexamethasone (Cy-Dex) (cyclophosphamide at a dose of 1000 mg/m(2) on Day 1 and dexamethasone at a dose of 40 mg per day on Days 1-4 and 9-12, repeated on Day 22) as initial therapy followed by stem cell mobilization, harvest, and finally ASCT. RESULTS: No significant difference was observed in the proportion of patients undergoing ASCT (VAD [86%] vs Cy-Dex [87%]). During the first 4 months after the initiation of therapy, the mortality rates were 5.8% for VAD and 1.9% for Cy-Dex (P = .08). The response rates after ASCT were comparable (partial response or better: VAD: 80% vs Cy-Dex: 81%). In both groups, the median event-free survival was 29 months, and the overall survival rate at 3 years was 75%. CONCLUSIONS: The current results indicated that Cy-Dex before ASCT has efficacy comparable to that of VAD. It also demonstrated that a short course of alkylator therapy using cyclophosphamide does not affect stem cell harvest or transplantation.

Udgivelsesdato: 2008 OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. MATERIAL AND METHODS: Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. RESULTS: FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. CONCLUSIONS: We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.