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BACKGROUND AND AIMS Belimumab (BEL), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with systemic lupus erythematosus (SLE) and a high level of disease activity or lupus nephritis (LN). BEL inhibits primary humoral immune responses by depleting naive B-cells that are dependent on BAFF for their survival while secondary humoral immune responses by memory B cells (MBCs) remain intact. Indeed, some studies reported an increase of circulating MBCs following neutralization of BAFF [1–3]. So far these effects of BEL on the MBC compartment in SLE patients have not been investigated. This study aimed to establish the dynamics of circulating MBCs in patients with SLE treated with BEL and to perform an in-depth analysis of the impact of BEL on the MBC compartment. METHOD First, extensive B cell subset phenotyping was performed prospectively by employing high-sensitivity flow cytometry (HSFC) based on EuroFlow protocols [4] in severe SLE/LN patients treated with BEL [5]. Additionally, in-depth characterisation of surging MBCs in circulation was performed by single-cell RNA sequencing (scRNA-seq). RESULTS HSFC established that the increase in MBCs was non-specific and observed in a broad range of MBC immunoglobulin subclasses peaking as early as 2 weeks after BEL initiation. Subsequent scRNA-seq analysis of the emerging MBCs revealed a non-proliferating phenotype with a prominent decrease in activation status. In these circulating MBCs, a large amount of migration and adhesion genes were downregulated suggesting that the accumulation of MBCs by BEL initiation was related to their impaired cell–cell adhesion disrupting cell-trafficking and preventing extravasation. CONCLUSION After initiation of BEL treatment, a substantial increase of circulating MBCs was firmly established in patients with SLE/LN. The surge of circulating MBCs appeared to be associated with disrupted lymphocyte trafficking of MBCs, thereby suggesting a new potential therapeutic mechanism of BEL on MBCs in SLE. These findings have important implications to our understanding and consequent improvement of B-cell targeted treatment strategies in patients with active SLE and LN as MBC accumulation in circulation might allow for more efficient targeting of the B-cell compartment.

Introduction: Colorectal cancer (CRC) is the third most common neoplasia in the world. Circulating tumor cells (CTC) have a prognostic value and can be useful in monitoring solid neoplasia. Only one method for CTC identification has received the approval and this is the CellSearch® system based on the immunomagnetic separation. Multiple markers are used in CTC identification, as epithelial markers and cytokeratines. CTC identification in peripheral blood is associated with a worse prognostic and reduced free survival in CRC. Material and methods: We performed a systematic search in PubMed database for articles that reports the circulating tumor cells in CRC until July 2019. We selected studies in English and French and the main words used for search were ‘circulating tumor cells’, ‘colorectal cancer’, ‘colon cancer’, ‘rectal cancer’, ‘flow cytometry’, ‘peripheral blood’. We included studies with more than 10 patients, where samples were collected from the blood in relation with surgery and flow cytometry was used as analyzing technique. Results: We included 7 studies in final analysis, that showed in flow cytometry analysis a cut-off value of CTC that can vary from 2-4 CTC/ 7.5 ml peripheral blood with a sensitivity of 50.8% and specificity of 95%. Patients with positive CTC were associated with higher T stage and positive lymph nodes, with a worse overall survival (OS) and disease free survival (DFS) comparing with negative patients. Conclusion: CTC are considered to be a prognostic factor who needs more validation studies in order to be included in the clinical practice.

Objective We aimed to evaluate the prognostic value of circulating tumor cells (CTCs) and the impact of intraoperative tumor manipulation on CTCs in colorectal cancer (CRC) patients. Methods We performed a prospective study on 40 patients with CRC stages I to IV who received curative surgery using the no-touch technique. Flow cytometry was used to identify CTCs in peripheral blood samples (4 mL/sample) collected at two surgical moments: skin incision (T1) and after surgical resection (T2). A threshold of ≥4 CTCs/4 mL blood was established for considering patients CTC positive. Results In the univariate analysis, CTC evaluation at T2 was correlated with female sex, vascular invasion, tumor localization in the colon and metastatic lymph nodes. In the multivariate analysis, only female sex and colon cancer maintained statistical significance. At a medium follow-up of 15 months (1–25 months), the mortality rate was 10% (n = 4), with no significant differences between the overall survival of T1 or T2 CTC-positive and CTC-negative patients. Conclusions Flow cytometry is a feasible CTC identification technique in CRC, and although surgical manipulation has no influence on CTC numbers, CTCs may serve as a prognostic and predictive factor.

The pathogeny of the metabolic syndrome (MetS ) is not fully elucidated, but a link between visceral obesity and the increase of the proinflammatory response was proven. Atherosclerosis, perceived as a metabolic complication, draws attention to the peroxisome proliferator-activated receptors- alpha (PPARα). PPARα receptors are transcription factors involved in lipid metabolism, inflammation and atheromatosis. Hence, it interferes in the pathogeny of cardiovascular diseases and other chronic diseases too (neurological, psychical, neoplasical). The study of the expression of PPARα and its modulation on different level may be beneficial in the treatment of metabolic syndrome, intervening in the modulation of another proinflammatory factors.

Background and Aims Belimumab (BLM), a recombinant human IgG-1λ monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active severe systemic lupus erythematosus (SLE) and lupus nephritis (LN). There is clinical evidence that combining belimumab with B cell depleting therapy can ameliorate disease activity in severe, refractory SLE patients. Although BLM is a B cell directed therapy and has been shown to significantly decrease total B cells, flow cytometry observations suggest a rapid increase of circulating memory B cells. The present study investigated the dynamics of B cell subsets in patients treated with or without BLM, with a special focus on the memory compartment in order to assess the characteristics of these memory B cells. Method We first performed extensive B cell subset phenotyping by high sensitivity flowcytometry according to the Euroflow protocol on whole blood from active lupus nephritis or SLE patients with other major organ involvement treated with standard of care (SOC) consisting of high dose steroids and mycophenolate mofetil combined with or without the addition of BLM. Next we characterized memory B cell gene expression profiles with single-cell RNA and V(D)J sequencing (ScRNA-SEQ). Results By employing high sensitivity flowcytometry, we established that the absolute increase in circulating memory B cells in SLE and LN patients was significant for patients who initiated BLM but not for patients treated with standard of care (Figure 1). The increase was observed in a broad range of memory B cell subsets (Unswitched, IgG1+, IgG2+, IgA1+, IgA2+) at 2 and 4 weeks following initiation of BLM treatment. This rise in memory B cells could hypothetically be attributed to either proliferation or homeostatic modulation of tissue-resident memory B cells causing a release into the circulation. ScRNA-SEQ cell cycle gene-expression was performed and established in both groups a non-proliferating phenotype [in approximately ∼94%] of memory B cells post-treatment, including absence of MKI67 as active proliferation marker. Additionally, BLM treatment did not result in an increased, and even in a severe reduction of the largest memory B cells clones after two weeks. In contrast, no change in clonality was seen after treatment with SOC. Together these data indicate that proliferation is not likely to be responsible for the observed increase in memory B cells by BLM. Furthermore, a clear difference was found in gene-expression levels between both treatment groups: BLM was responsible for the upregulation of 72 vs 10 genes in SOC, likewise 162 vs 32 genes were downregulated. Most importantly, a significant downregulation of the migration genes SELL (CD62L), CCR7, ITGB1, RAC2 and ICAM2, were specifically seen in BLM treated patients, possibly reflecting disrupted lymphocyte trafficking preventing memory B cells to either transmigrate into tissue or be retained at the tissue level. Conclusion The addition of BLM compared to standard of care in SLE patients leads to an increase of memory B cells in the circulation which appeared independent of proliferation. The process was accompanied with a strong modulation of gene-expression levels including a reduced expression of migration markers pointing towards disrupted lymphocyte trafficking. Altogether, these data could have important implications to further improve treatment strategies in severe SLE or lupus nephritis patients, for instance by establishing a deeper depletion of (autoreactive) memory B cells by the addition of rituximab after the initiation of belimumab.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates

A primigravid woman with Covid-19 related respiratory insufficiency was admitted into a tertiary Intensive Care Unit at 23 3/7 weeks’ gestation. Highly sensitive flow cytometry of peripheral leukocytes indicated significantly suppressed naïve T- and B-cell compartments. The suppressed immune cell responses led us keep the initially started administration of corticosteroids for fetal and maternal indication at a low dose. After three weeks her B-cell response peaked, SARS-CoV-2 was cleared and clinical improvement ensued a week later. At 28 weeks’ gestation, a son of 1570 g was born by cesarean section. She was extubated two days postpartum and discharged from hospital 5.5 weeks postpartum.

Acute Myeloid Leukaemia (AML) is a neoplasia characterised by rapid proliferation and an increased rate of relapses. The AML blasts display features of antigen-presenting cells (APC), and thus can directly modulate the anti-tumour T cell responses. The bone marrow of a group consisting of 30 newly diagnosed patients and four healthy donors (HD) was investigated for the expression of HLA-DR, several molecules involved in MHC-II antigen-presentation and MHC-II groove editing, like HLA-DM, CD74 and CLIP, as well as a set of immune checkpoint ligands, like ICOS-L, B7.2, PD-L2 and B7-H3. The patients were further characterised for their genetic anomalies and distributed to favourable, intermediate and adverse ELN risk categories. We were able to show that while 23% of our patients displayed a low level of HLA-DR surface expression, all patients displayed higher HLA-DM and CD74 expression compared to HD. However, a higher CLIP expression was noticed only in the HLA-DR low patients. The co-inhibitory PD-L2 and B7-H3 molecules were increased in the cases with normal HLA-DR expression; oppositely, the co-stimulatory ICOS-L and the dual function B7.2 were significantly increased in the cases with HLA-DR low expression. Furthermore, no favourable ELN risk cases were found within the HLA-DR low group. All in all, these data show that the AML with low versus normal HLA-DR expression display different profiles of MHC class II machinery molecules and B7 ligands, which are correlated with distinct ELN stratification. Furthermore, as our study included healthy individuals, it offers valuable information about the expression levels that should be considered as normal for these markers known to cause differences in peptide repertoires, reflected further in distinct T-cells polarisation pathways.

Background: Acute basophilic leukemia is a rare subtype of acute myeloid leukemia, as categorized by the 2008 World Health Organization classification of myeloid neoplasms. Acute basophilic leukemia diagnosis requires thorough morphological, cytochemical, immunophenotypic, molecular, and cytogenetic studies and exclusion of other hematological neoplasms associating basophilia. The disease course is defined by histamine driven, occasionally life-threatening respiratory, cardiovascular, cutaneous or digestive complications, as well as primary refractoriness to standard therapy. Clinical presentation: We herein report a case of a 63-year-old asthmatic female patient diagnosed with acute basophilic leukemia, associated with previously unpublished cytogenetic features and FLT-3 ITD mutation, pulmonary leukostasis and spontaneous pulmonary capillary leak syndrome, which worsened immediately following chemotherapy initiation. Respiratory complications were successfully managed, but recrudesced upon emergence of refractory disease and were ultimately fatal. We highlight the likelihood of pulmonary complications induced by basophil degranulation and tumor lysis in hypercellular acute basophilic leukemia and the potential benefit of histamine receptor blockade in this setting.

Background:Belimumab (BLM), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active severe systemic lupus erythematosus (SLE) and lupus nephritis (LN). There is clinical evidence that combining BLM with B cell depleting therapy can ameliorate disease activity in severe, refractory SLE patients1. Although BLM is a B cell directed therapy and has been shown to significantly decrease total B cells, flow cytometry observations suggest a rapid increase of circulating memory B cells (MBC)2.Objectives:To investigate dynamics of B-cell subsets in SLE patients treated with or without BLM, with a focus on assessing MBC characteristics.Methods:Extensive B cell subset phenotyping was performed by high-sensitivity (HS) flow cytometry (acquisition of 107 leukocytes; per EuroFlow protocols3) on samples from active LN or SLE patients with major organ involvement treated with standard of care (SOC) consisting of high dose steroids and mycophenolate mofetil combined with or without the addition of BLM. MBC gene expression profiles were characterized with single-cell RNA and V(D)J sequencing (ScRNA-SEQ).Results:By employing HS flowcytometry, we established that the absolute increase in circulating MBC in SLE and LN patients was significant for patients who initiated BLM (Figure 1). The increase was observed in a broad range of MBC subsets (Unswitched, IgG1+, IgG2+, IgA1+, IgA2+) at 2 and 4 weeks following initiation of BLM treatment. This rise in MBC could hypothetically be attributed to either proliferation of blood MBC, BLM induced migration of tissue-resident MBCs or BLM related retention of tissue-destined MBC in the blood. ScRNA-SEQ analysis of cell cycle gene-expression was performed and established in both groups a non-proliferating phenotype [in approximately ~94%] of MBC post-treatment, including absence of MKI67 as active proliferation marker. Clonal diversity analysis comparing week 2 with baseline revealed an unexpected decrease of the largest MBC clones in BLM, whereas no change in clonality was observed with SOC alone. Together these data indicate that proliferation is unlikely to be responsible for the observed increase in MBC by BLM. Furthermore, a clear difference was found in gene-expression levels between both treatment groups: BLM was responsible for the upregulation of 72 vs 10 genes in SOC, likewise 162 vs 32 genes were downregulated. Most importantly, a significant downregulation of the migration genes SELL (CD62L), CCR7, ITGB1, RAC2 and ICAM2, were specifically seen in BLM treated patients. This may reflect disrupted lymphocyte trafficking, preventing MBCs from transmigrating from the blood into tissue owing to reduced migration molecules, or preventing MBCs from being retained at the tissue level owing to reduction in tissue adhesion proteins.Conclusion:The addition of BLM to SOC significantly increases MBCs in patients with SLE independently of proliferation, accompanied by a strong modulation of gene expression, including reduced expression of migration markers pointing towards disrupted lymphocyte trafficking. These data may have important implications for improving treatment strategies in patients with LN or severe SLE, as a deeper depletion of autoreactive MBCs could be established by adding B-cell-depleting therapy after the initiation of BLM.Figure 1.Change in pre-germinal center and memory B cell counts from baseline to week 4 of patients with SLE or LN treated with SOC (n=8) or SOC+BLM (n=11).References:[1]Arends EJ et al. Long-term effects of combined B cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results. Nephrol Dial Transplant. 2020 Jun 27 gfaa117.[2]Stohl W et al. Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with SLE. Arthritis Rheum. 2012;64(7):2328-2337.[3]Blanco et al, Age-associated distribution of B and plasma cell subsets in peripheral blood - J Allergy Clin Immunol 2018 141 2208-2219.Disclosure of Interests:Eline J. Arends: None declared, Mihaela Zlei: None declared, Christopher M. Tipton: None declared, Zgjim Osmani: None declared, Sylvia Kamerling: None declared, Ton Rabelink: None declared, Ignacio Sanz: None declared, Jacques J.M. van Dongen Paid instructor for: BD Biosciences: Educational Services (fees for LUMC), Consultant of: BD Biosciences and Cytognos (fees for LUMC), Grant/research support from: GSK (flow cytometry studies for GSK BLISS-BELIEVE study NCT03312907), Cees van Kooten: None declared, Y.K. Onno Teng Consultant of: Aurinia provided financial compensation for consultancy, Grant/research support from: GSK provided belimumab for free for the Synbiose-2 clinical trial and provided an unrestricted grant to conduct the study.

Introduction. Several alternative methods to peripheral blood DNA extraction have been implemented so far. Saliva seems to represent a very advantageous type of sample, easy to harvest and able to generate DNA yields comparable to those extracted from blood mononuclear cells. Material and methods. 8 patients suspected of ankylosing spondylitis, 9 patients with various hematological malignancies, displaying post-chemotherapy leucopenia and 30 healthy volunteers were included in our study. DNA was extracted with various commercially available kits and used for HLA typing either by PCR amplification, or by PCR followed by hybridization. Results. Our data regarding HLA typing support already published results regarding the good DNA quality that allows its use in various molecular biology techniques. However, when attempting to use saliva from immunosuppressed patients for DNA extraction we have generated very low yields, comparable again with the ones obtained from peripheral blood. Flow cytometry and immunocytochemistry investigations confirmed the low number of leukocytes present in the saliva of these patients, while the number of epithelial cells was virtually unchanged. Conclusions. The main source of saliva DNA seems to be represented by leukocytes present in this fluid and not by the epithelial cells. Under these circumstances, for immunosuppressed patients saliva cannot represent an alternative to blood when attempting DNA extraction.

Current management of acute myeloblastic leukemia (AML) involves induction chemotherapy followed by risk stratified consolidation approaches, consisting of high dose chemotherapy or allogeneic stem cell transplantation. AML patients are at high risk of invasive fungal disease, particularly mold infections, due to the leukemia and chemotherapy related immune deficit. We present an AML patient with prolonged fever and neutropenia after induction therapy, during which she developed concomitant Candida zeylanoides bloodstream infection and invasive pulmonary aspergillosis. The response to Caspofungin and Voriconazole therapy was delayed, and this dictated the surgical management of the remnant pulmonary lesions. A histological confirmation of aspergillosis was thus obtained. Evaluation of host risk factors for invasive fungal disease, prompt scale-up of the diagnosis scheme and initiation of antifungal therapy are mandatory in order to ensure patient survival.

Introduction: Since the introduction of novel agents into the treatment of Multiple Myeloma (MM), much deeper remissions can be achieved. This leads to a demand for disease evaluation tools with higher sensitivity and clinical practicability. In various clinical trials the assessment of the MRD status has been proven to have strongest prognostic value for MM patients (pts). The Euroflow Consortium has pioneered a highly standardized next generation flow (NGF) approach with the establishment of two 8-color tubes tests (Flores-Montero 2017). However, due to cost-intensive and time-consuming preconditions, this MRD approach might not be feasible in every center. Thus, other readily available, validated MRD tools may also guarantee highly sensitive MRD evaluation which allows for early relapse detection and to steer treatment, such as decision upon need for consolidation, duration of maintenance, therapy pauses or even earlier FDA/EMA approval of novel antimyeloma agents. Methods: Our 10 color single tube MFC panel consisting of the antigens CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117 and CD200, was systematically validated using Fluorescence-minus-one controls, consecutive spike-in-controls, consistency analyses and measurement of 9 MM cell lines. Thereafter, we assessed 131 Bone Marrow (BM), peripheral blood (PB), and leukapheresis (LA) samples from 97 different MM, MGUS and SMM pts and 13 healthy individuals (HI) as controls. Samples were processed within 6-20 hours of aspiration and a bulk lysis was performed. Subsequently, >3x106 viable nucleated cells were acquired on a BD LRSFortessa Cytometer. Data analysis was performed using the BeckmanCoulter software Kaluza. BM was sampled at initial diagnosis (ID) or at progression (PD), after standard therapy (ST) or stem cell transplantation (SCT). The study was performed with written consent from all pts and approval from the ethics committee. Results: Our MFC approach confirmed the antigen expression in all MM cell lines (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2) as previously reported. The spike-in-controls determined the limit of detection (LOD) at 10-5. This LOD was achieved in 89% of our MRD samples. An easy-to-adapt gating strategy to identify abnormal plasma cells (aPC) vs. normal plasma cells (nPC) was also established in our MM pt samples. In this cohort, we identified 10 distinct aPC and nPC subpopulations that differed in their expression pattern, suggesting clonal subtypes of MM. Moreover, in 19% of BM samples, two malignant subpopulations coexisting in the same pt, were identified. Since our BM cohort consisted of 46% samples at ID or PD vs. 54% posttreatment samples after ST and SCT, clonality variation in the former, differences of aPCs vs. nPCs ratio and to the latter group could be determined. Moreover, significant differences of much higher percentage of aPC in symptomatic MM vs SMM/MGUS pts were readily verified with our panel (p=0.0087). With a median time of 51 days from treatment to MRD evaluation, 24% of post treatment samples were MRD negative (MRD-) at the 10-5 level ( Since we also assessed pre- and post-treatment samples, antibody expression levels on PC from ID to post treatment demonstrated a significant normalization in Median Fluorescence Intensity (MFI) of 5 antibodies (CD56, CD81, CD45, CD200, CD27). These may thus serve as informative markers for the MRD assessment of these samples. Conversely, with progression from remission to PD, CD81 and CD45 antibodies showed significant decreases (p=0.0007 and p=0.0026, respectively) in expression, which suggest that both may serve as early relapse markers. Of the assessed LA samples at harvest before ASCT, 44% were MRD-, warranting future investigations into possible implications for pts' prognosis, treatment and PFS/OS data. Conclusion: Here we present a readily available, cost-effective, quick and highly validated MFC panel with an easy to adapt gating strategy that allows for precise aPC vs. nPC assessment in- and outside clinical trials. Our 10 color MFC panel is applicable in BM samples of MM pts and precursor diseases, as well as in LA samples. With the additional MFC information, ideally at ID and repeatedly after treatment, readily available individual treatment decisions seem possible to obtain for every MM patient. Disclosures Wäsch: Amgen: Other: travel, Research Funding; Sanofi: Other: Travel, Research Funding; Jazz: Other: travel, Research Funding; Celgene: Other: travel, Research Funding; Gilead: Other: travel, Research Funding; Takeda: Consultancy; Gilead: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

The hallmark of chronic myeloid leukemia is the existence of the cytogenetic evidence of the Phyladelphia chromosome (reciprocal translocation between chromosome 9 and 22, and is specifically designated t(9;22)(q34;q11). The result of the translocation is the oncogenic BCR-ABL gene fusion, located on the shorter derivative 22 chromosome. This gene encodes the Bcr-abl fusion protein the BCR-ABL tyrosine kinase - a protein that is continuously activated. The result of this unregulated activation is an unregulated neoplastic type cell division - chronic myeloid leukemia. The first targeted molecular treatment operating in cancer- inhibitor of the BCR-ABL tyrosine kinase, Imatinib mesylate, is the standard of care for chronic myeloid leukemia (CML) Methods. A retrospective review of patients in one department of hematology with a diagnosis of Ph/BCR-ABL positive CML and received imatinib. Results. From 2002 to 2012, 66 patients in CML-CP received imatinib were introduced in the study. 22 (33%) patients received imatinib as upfront therapy, the others as second or third line treatment. The cytogenetic response (CyR) achieved was major in 62% with 56% complete cytogenetic response (CCR), no CyR in 17 patients (25%). The molecular response was complete in 13 (20%) and major in 16 (24%) patients. Better cytogenetic and molecular responses were achieved by those with low and intermediary risk (Sokal) Seven patients developed under imatinib additional cytogenetic anomalies: supplemental chromosome 8 (6), duplication of Ph1 (2), trisomy 17 and 19 (1). The median of follow-up was 69 months (range 18-180) and under imatinib was 52 months (range 3-126). The Sokal score was a better predictor of survival than Hasford’s. Conclusions. Imatinib remains the best first line treatment, but there are still a significant number of patients who did not achieve a CyR. The responses and survival were not influenced by the previous treatments but the earlier introduction of imatinib is better. The Sokal score seems to have a better prognostic role. The survival of patients with CML is evidently improuved by tyrosin kynase inhibitors.

The clinical management of severe pathological conditions, such as B-cell chronic lymphocytic leukemia (B-CLL), is subject to continuous optimization and re-evaluation. Patients may fully benefit from rapid, standardized laboratory tools designed to facilitate their early stratification according to disease risk, stage and prognosis. Such technologies may also aid the clinician in selecting the therapeutic option with the greatest chances of success. The presence of specific genetic abnormalities are frequently associated with the clinical outcome of oncologic patients in general, and B-CLL patients in particular. In the current study, a group of 58 B-CLL patients were evaluated for the detection of gene copy number alterations (deletions or duplication/ amplifications) within 45 distinct genetic targets, by means of a novel molecular methodology, Multiplex Ligation - Dependent Probe Amplification (MLPA). Simple or complex genetic defects were identified in 67% of cases, and the most common aberrations observed were: deletion of the short arm of chromosome 13 in 33% of cases, deletion of the long arm of chromosome 11 in 16% of cases, trisomy 12 in 16% of cases, and deletion of the short arm of chromosome 17 in 7% of cases. The main conclusion of the study presented here points towards MLPA as a potential key step of clinical management protocols in B-CLL, providing that it will be fully standardised for routine diagnosis.

Obiectiv: Limfocitele T se constituie în factori celulari importanţi ai răspunsului imun. Leucemia limfocitară cronică B se caracterizează prin defecte ale sistemului imun. Studiul nostru îşi propune caracterizarea diferitelor subseturi maturative şi funcţionale limfoide T şi NK la pacienţi aflaţi în stadii distincte de boală. Material şi metoda: au fost utilizate celule mononucleate separate în gradient de densitate de la 43 de pacienţi cu LLC-B (16 femei, 27 bărbaţi, vârsta 68±10 ani, 8 în Rai0, 22 în Rai1/2 şi 13 în Rai3/4) în vederea analizei prin citometrie in flux multiparametrică. Au fost identificate şi comparate între diferite stadii Rai, subseturile limfoide T CD4+ (naive, memorie centrală, memorie periferică/efector, reglatorii, foliculare-TFH, CXCR3 şi/sau CCR4+), CD8+ (naive+memorie, efectorii, senescente) şi NK (CD57 şi/sau CD94+). Rezultate: Limfocitele T totale circulante se găsesc în numere crescute la pacienţi în stadii avansate ale bolii. Majoritatea limfocitelor T CD4+ sunt celule ce au întâlnit antigenul, cu subseturi crescute de tip reglator, folicular şi CXCR3+ (asociat profilului Th1). Expansiunea limfocitelor citotoxice CD8+ este corelată cu un subset diferenţiat terminal, senescent, CD57+. În ceea ce priveşte subseturile limfocitare NK, nu s-au identificat diferente semnificative între diferite stadii ale bolii. Concluzii: Comportamentul leucemiei limfocitare cronice B pare a fi asociat cu o creştere în valori absolute a limfocitelor T CD4+ reglatorii şi foliculare circulante, dar şi cu expansiunea limfocitelor T citotoxice senescente. Modularea răspunsului T la pacienţii cu LLC-B joacă un rol important în evoluţia bolii şi se constituie în eveniment cheie al compensarii imunodeficientei caracteristice stadiilor avansate ale bolii.

Introduction: MM is characterized by the accumulation of aberrant BM plasma cells (aPCs). The use of novel agents for anti-MM treatment has enabled the achievement of deeper responses and prolonged progression free survival (PFS) and overall survival (OS). These advances have created the need for sensitive means to detect residual PCs. MFC allows aPC detection with high sensitivity and is used with NGS in clinical trials (CTs) (Paiva, 2015-2018). It allows the detection of residual cells after anti-MM therapies and is a risk denominator of pt subgroups and post-therapeutic outcome. The incorporation of cost-effective, readily available and standardized MRD tools into CTs and clinical practice seems a key requisite. It may allow the improvement of clinical decisions: 1) timing of stem cell transplantation (SCT), 2) need for consolidation, 3) duration of maintenance, 4) therapy modulations and 5) early relapse detection. Since MRD gains importance for novel agents' potency to achieve MRD negativity, including FDA/EMA-approvals, we assessed MFC for detection of aPCs, their change to treatment and over time. Methods: We systematically validated a 6-, 8- and 10-color panel using CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117, CD200 and kappa/lambda (k/l) in 9 different MM cell lines (MMCLs) and 213 BM, PB, leukapheresis (LA), extramedullary (EM) samples of 122 different MM pts and 14 healthy individuals (HI). These were immediately analyzed after sampling, using a bulk-lysis protocol. To reach a high sensitivity, >3x106 viable nucleated cells were analyzed. MFC validation included fluorescence-minus-one-(FMO), isotype- and spike-in-controls to evaluate the reliability and sensitivity of our panels. Spike-in controls were performed using a dilution assay to recover defined MMCL cells in the PB of HI. Data analysis was accomplished using the BeckmanCoulter software Kaluza®. We compared MFC using BM and PB samples of HI, MM pts at initial diagnosis (ID) or with progression (PD), after standard therapy (ST) and SCT. Additionally, BM samples at ID were sorted into each 4 aPC- and normal PC- (nPC) subpopulations for DNA and RNA sequencing and follow-up. The study was performed with written consent of all pts and HIs and approval of the ethics committee. Results: The antigen expression levels in 9 MMCLs using our 6-, 8- and 10-color panel could be confirmed as reported. The expression levels of antigens included in all panels were similar in all MMCLs (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2). For all panels, we established easy to adapt gating strategies, using commercially available software to identify aPCs vs. nPCs in all analyzed samples. Using the 6-color panel, differentiation into up to 4 phenotypic subpopulations of aPCs and nPCs in MM pts and up to 4 subpopulations of nPCs in HI was achieved, depending on the heterogeneous phenotype the individual was expressing. Moreover, both DNA and RNA of obtained subpopulations were collected and analyzed via sequencing. Since in 1% of our samples, aPCs remained undetected via 6-color panel using surface antigens exclusively, we added k/l and did detect aPCs in all pts. The 10-color panel identified aPC- and nPC-subpopulations in even more detail. We determined a MRD sensitivity of 10-5, confirmed via independent dilution assays. In 48% of BM and PB samples, aPCs were determined at ID and PD; 52% were assessed for MRD after ST and SCT. Of the latter, 55% were BM samples, of which 5% showed MRD negativity of Conclusion: Our 6-, 8-, and 10-color panels allow highly sensitive detection of aPCs vs. nPCs in MM samples. These panels have been validated using various controls, dilution assays, 9 MMCLs, MM pt samples and HI and can be implemented into routine diagnostics. The analysis of DNA and RNA expression patterns in distinct subpopulations will generate relevant insight, which subclones contribute to response vs. resistance mechanisms. The EuroFlow Consortium have established a two 8-color tubes test for identification of aPCs, which - albeit used worldwide - may not be applicable for every center and every pt outside CTs. Therefore, meticulously validated alternatives as shown here are of interest. Our 8-color panel has been included into our CT portfolio. Disclosures Azab: Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding. Zeiser:Jazz Pharmaceuticals: Research Funding. Wäsch:Pfizer: Honoraria.

Multiple myeloma, also called Kahler disease or myelomatosis is a debilitating and incurable malignancy characterized by proliferation of malignant plasma cells and an increased production of monoclonal paraproteins. In multiple myeloma, the abnormal proliferation of plasma cells within the bone marrow interferes with the production of blood cells therefor leading to anemia, leukopenia and thrombocytopenia. Another characteristic of disease is the activation of osteoclasts which leads to osteolytic lesions accompanied by fractures, bone pain, hypercalcemia and renal failure. Since a key factor in the pathology of multiple myeloma is represented by the interaction between bone marrow stroma and plasma cells we have designed an in vitro experiment where L363 myeloma cells have been grown together with bone marrow stromal cells. The negative control was represented by L363 cells cultured alone. Following coculture, RNA from L363 plasma cells was extracted, revers-transcribed and analyzed by microarray techniques to identify biological processes and pathways which are affected by differentially expressed genes. Among these biological processes we mention regulation of cell cycle, apoptosis, and STAT genes activation.

Objective To develop an in vitro culture system for rapid assessment of multiple myeloma (MM) cell growth. Methods MM cells lines (MMCLs) L363, U266, and RPMI-8226, and bone marrow (BM)–derived plasma cells (PCs) from MM patients were evaluated for their in vitro growth using various stroma (BMSC, M210-B4, and osteoclasts [OCs]) and cytokine support combinations (combination A: interleukin [IL]-6, vascular endothelial growth factor, insulin-like growth factor-1 vs combination B: A plus hepatocyte growth factor, IL-13 vs combination C: IL-6, insulin-like growth factor -1, stromal-derived growth factor-1, Galectin-1, IL-1α). Results We found a significant effect of stroma, notably affecting growth of L363 cells. Cytokine combination A had the highest growth impact, whereas B and C were of lesser benefit. The contribution of combined cytokines and stroma for MMCL growth was moderate and the viability of MMCLs was best preserved with OCs. One of the most commonly used PC-marker CD138, expressed on all MMCLs on day 0, showed a gradual downmodulation upon all culture conditions, possibly induced as a stroma-triggered phenotypic change, and leading to the ability of MM cells to dedifferentiate into immature, resilient phenotypes. PC-enriched BM samples from 7 of 10 MM patients could be maintained in culture, again profiting from stroma more than cytokines alone. Conclusions Our data demonstrate a consistent growth advantage provided by BMSCs on MMCLs and primary MM cells, preserved viability with OCs, and phenotypic and morphologic heterogeneity of primary MM cells during culture. Further identification of key components involved in MM cell growth, coupled with our understanding of drug sensitivity offers the potential to better define the disease pathogenesis and to identify novel therapeutic targets.

We report a successful ongoing pregnancy obtained in a case of total globozoospermia after intracytoplasmic morphologically selected sperm injection (IMSI) with oocyte activation. The first semen analysis on investigation showed partial globozoospermia. However, under high magnification assessment at oocyte retrieval only round headed sperm were observed. Considering the high risk of a complete failure to fertilize from IMSI the couple gave written informed consent to the use of oocyte activation media post IMSI. One embryo fertilized, developed to a hatching blastocyst and was transferred resulting in an ongoing pregnancy. This successful outcome shows the use of IMSI is useful in the evaluation of total globozooozpermia and therefore aids in the justification of the use of oocyte activation media. Electronic supplementary material The online version of this article (doi:10.1186/s12978-015-0031-x) contains supplementary material, which is available to authorized users.

The coexistence of t(9;22) and inv(16) has been described in a very limited number of cases of CML, de novo or therapy-related AML. We report a patient with CML who presented both inversion of chromosome 16 and Philadelphia chromosome and evolved towards the blast phase under treatment with Imatinib. Laboratory diagnosis and monitoring was made by flow cytometry, conventional cytogenetics and molecular genetics techniques. The inv(16), detected by karyotyping in the Philadelphia chromosome positive clone at the moment of the blast transformation, was retrospectively assessed by means of real-time PCR, and was proved to have been present since diagnosis. The bone marrow biopsy performed in the blast phase of CML confirmed the presence of blasts belonging to the myeloid lineage, with indications of monocytic differentiation, frequently associated with inv(16). Moreover, the case also associated a F359V tyrosine kinase domain mutation, resulting in intermediate resistance to Imatinib and Nilotinib, which imposed therapy-switch to Dasatinib. In our case the evolution was progressive, followed by death due to lack of response to tyrosine kinase inhibitors, 18 months after diagnosis. The coexistence of t(9;22) and inv(16) in CML seems to be associated with an aggressive clinical evolution and resistance to tyrosine kinase inhibitor therapy. Due to the very small number of cases described in literature, therapeutic decisions are still difficult for patients displaying these abnormalities

Introduction. Metabolic syndrome (MS) is a cluster of distinct metabolic alterations with an increased cardiovascular risk. Peroxisome Proliferator-Activated Receptor - Alpha (PPARα), member of the nuclear receptor superfamily of transcription factors, is critically involved in the management of lipid metabolism during homeostasis or inflammatory stresses in various cell types and represents one of the therapeutic targets in MS. We analysed the PPARα expression in leukocytes of pacients with MS, in order to address PPARα involvement in these group of diseases. Material and method. Our study included 57 adult patients recruited under informed voluntary consent, investigated in order to establish whether they present MS, according to International Diabetes Federation (IDF) European guidelines and grouped in 2 lots: the MS Lot (26 patients) and control group, non-MS Lot (31 subjects). Common clinical and laboratory parameters targeted in MS evaluation were determined for all the studied cases. The expression levels of 2 molecules, PPARα and CD36 were evaluated in various circulating leukocyte populations of these patients by an optimized flow cytometry method. Statistic analysis clarifying the significance of value differences for various parameters measured was performed under SPSS and simple statistical tests (Pearson, t-Student, Chi -test). Results and discussion. The fluorescence staining for PPARα were significantly dimmer when comparing the cellular expression in eosinophils (p

Traffic of tumorand normal cells through the peripheral blood (PB) of patients with B-cell chronic lymphocytic leukemia (B-CLL) to the lymph nodes (LN) or spleen/ liver sites is governed by specific changes in surface and intracellular molecule expression. The study aims to investigate the potential association between different lymphocyte subsets, chemokine receptors or genetic alterations and specific clinical signs in a group of B-CLL patients. Forty-three patients were included in the study. The expression of CCR7, CXCR5, CXCR3, CCR4, CD3, CD4, CD8, CD27, CD28, CD45RA, CD25, CD127, CD38 was tested by multiparameter flow cytometry. Genetic alterations were determined by MLPA. We found increased frequency of CD38+ B-CLL cells directly correlated with the presence of LN>5cm. CXCR5 and CCR7 are homogenously expressed by monoclonal B-CLL cells. CCR4+ B-CLL cell frequency is found to be lower in the PB of patients presenting particular LN involvement. Heterogeneous and complex genetic alterations were found and only the presence of trisomy 12 associated with less frequent axillary LN involvement. We also report a significant increase in the frequency of total T cells and T cell subsets (effectorand central memory CD4+ T cells, regulatory T cells, follicular T helper cells, distinct functional CD8+ T cells) with the occurrence of specific clinical manifestations. Chemokine receptor expression on circulating CD4+ T cell subsets was augmented in connection to some specific LN locations. Consequently, clinical manifestations in B-CLL are linked to both, factors intrinsic to the monoclonal B cells, and external influences coming from the microenvironment.

Introducere. Pacienţii cu leucemie acuta limfoblastică (LAL) neonatală prezintă în mod frecvent, o tran- slocaţie particulară, t(4;ll)(q21;q23), care duce la fuziunea genei MLL (mixed lineage leukemia) de pe cromo- zomul 11 cu gena AF4 de pe cromozomul 4. Deşi în literatura de specialitate au fost descrise anterior cazuri de LAL neonatală caracterizate prin prezenţa unor produşi de fuziune MLL-AF4 cu variate puncte de ruptura, cazul prezentat în acest material este inedit, atât prin complexitatea fuzatului MLL-AF4 identificat, cât şi prin particu- larităţile sale clinico-biologice. Materiale şi metode. Intr-o probă de sânge periferic provenită de la un nou-năs- cut în vârstă de 21 de zile a fost testată, prin biologie moleculară, prezenţa genelor de fuziune: TEL-AML1, BCR-ABL(plQO). E2A-PBX1 si MLL-AF4. Fragmentul MLL-AF4 identificat, având o lungime neobişnuită, a fost purificat şi secvenţiat. Evaluarea imunofenotipică a blaştilor leucemici circulanţi s-a efectuat prin citometrie în flux, multiparametrică, utilizându-se un citometru FACSCanto-II (Becton-Dickinson). Rezultate. S-a identificat un transcript de fuziune MLL-AF4 cu o secvenţă particulară (fuziunea exonului 12 de pe gena MLL cu exonul 4 de pe gena AF4). Evaluarea imunofenotipică a evidenţiat, de asemenea, o evoluţie particulară: la diagnostic s-a des- cris o clonă malignă cu fenotip de precursor limfoid B, în timp ce la recădere s-a evidenţiat expansiunea predominantă a unei clone monocitoide. Prezenţa aceluiaşi transcript MLL-AF4(el2-e4) a fost evidenţiată şi la recădere, în absenţa oricăror altor fuzaţi caracteristici pentru lineajul mieloid. Concluzie. Considerăm că particularităţile punctelor de ruptură care au dus la generarea rearanjamentului MLL-AF4 în acest caz sunt inedite, nemaifiind menţionate anterior în literatura de specialitate. Detecţia sa timpurie are potenţial predictiv pentru switch-ul imunofenotipic şi implicaţii în optimizarea strategiilor terapeutice.

Bone marrow mesenchymal stem cells are important for both research and clinical purpose. A number of culture methods for these cells are available on the market, many of them consisting of specialized growing media in combination with growth factors. Our goal was to optimize a less expensive culture method for bone marrow mesenchymal cells.Eight samples of bone marrow aspirates from patients were used. Out these 8 samples 2 were from healthy people, 3 from chronic granulocytic leukemia patients, 2 from multiple myeloma patients and 2 from patients with myelodysplastic syndrome. Bone aspirates from healthy people were used to optimize the culture method and the rest were used for testing the optimized method. Two methods were tried: 1. Cell culture starting from whole bone marrow, 2) cell culture after bone marrow separation in density gradient with Histopaque.Cell culture starting from whole bone marrow gives better yields for mesenchymal stem cells than methods which include gradient density separation of mononuclear cells with Ficoll-Histopaque.We have optimised a less expensive cell culture method for bone marrow mesenchymal cells.

The objective of this study was to determine the relationship between the activity of the enzyme aspartate aminotransferase (AST) and IL1-beta in gingival crevicular fluid (GCF) on animal model with experimentally induced diabetes mellitus and periodontal disease.During our study we used 15 Wistar rats, divided into three groups: I--control, II--with experimental model of periodontal disease, III--with experimental model of periodontal disease and diabetes. The sampling of GCF was realized using Whatman no. 1 paper strips introduced in the gingival sulci from mandibular left and right molars. For the determination of AST in GCF we used a spectrophotometric method while gingival fluid IL-1beta determinations were realized through immune enzymatic methods, using an ELISA kit (rlL-1beta).The results displayed 3.47 times increased gingival fluid AST values in the stimulated experimental model (with periodontal alteration) when compared to control values (without periodontal disease), while in diabetes an increase of 6.139 times higher compared to control (without periodontal disease) were recorded. Moreover, the levels of periodontal disorder-induced IL-1beta were 3.54 times higher compared to control (group II--218.490 pg/mL vs group I--61.691 pg/mL), the most significant increase being recorded for the group with diabetes associated to periodontitis (492.129 pg/mL).The present study found a high level of agreement between the presence of AST and IL-1beta in GCF in the experimental model of diabetes associated to periodontal disease, elevated when compared with the periodontal disease only model, and both higher when compared to control group.

Leukemic cells have unique aberrant phenotypes, which permit identification of this cells at diagnose and in evolution of the disease. Signaling molecules with other cells and bone marrow stroma are part of the leukemic cells phenotype. Genetic and molecular abnormalities have the main prognostic significance and confer the leukemic cell status. The main aim of the current study is to identify correlation between recognized prognostic factors in acute myeloid leukemia (AML) patients and other phenotypic markers.Imunophenotypic analysis (BDFACS CantoII, FACSDiva Software) was performed on peripheral blood/bone marrow aspirate samples of 56 patients diagnosed with AML (9 M0, 3 M1, 10 M2, 4 M3, 28 M4/M5, 1 M6, 1 M7) between 2007-2009 in Hematology Department of "Sf. Spiridon" Hospital Iaşi. We used an extended panel of monoclonal antibodies and we determined the level of expression of cytokines receptors (IL3Ra, IL7R) and chemokines (CXCR4, CKR5).In our study, IL7R expression on AML blasts was significant correlate with low WBC count at diagnosis (p = 0.04) and with multilinear displasia (p = 0.01), high CXCR4 expression was correlate (p = 0.05) with lack of response at first induction therapy and CD123 (IL3Ra) expression was correlate with M4 FAB phenotype. Survival was negative influenced by presence of IL3R on AML blasts, but flt3 mutations, CXCR4, IL7R expression on leukemic cells, other phenotypic aberrancies did not influenced treatment response and survival in our patients population.Complete investigation of leukemic cells phenotype extended with cytokines/chemokines receptors at diagnostic is useful for correct characterization of the disease, for discover new prognostic categories and for better identification of minimal residual disease.

Bone marrow stromal cells (BMSCs) have been found to support leukemic cell survival; however, the mechanisms responsible are far from being elucidated yet. The main aim of the current study is to identify particular cytokine/chemokine patterns of acute myeloid leukemia (AML) cells, and, on a longer term, to correlate them with the patient outcome and response to therapy. Therefore, the influence of BMSCs on in vitro modulation of cytokine secretion (IL-1beta, TNF-alpha, IL-10, IFN-gamma, IL-4, IL-5, and IL-2) by AML cells as well as the AML cells supportive capacity of BMSCs-derived soluble factors was investigated.With this purpose, we used an in vitro experimental model consisting in the evaluation of the effect of BMSC confluent layers-conditioning medium (BMSC-CM) on M4/5 AML cell cultures.Our results show that BMSC-CM from both AML patients and healthy subjects conferred a substantial beneficial effect on AML cells throughout the culture (p=0.0002 and 0.0020 respectively at 24 hours and p=0,0013 and 0,0030 respectively at 72 hours), with a temporary increase in AML cell viability conferred by BM plasma from AML patients. Significant differences were observed with respect to IL1 b secretion which was upregulated in AML cell cultures both after 24 and 72 hours following the addition of AML-BMSC-CM, in contrast to control-BMSC-CM.Our results suggest the contribution of BMSCs from AML patients to the generation of particular factors which may be key players involved in the in vivo maintenance of the malignant clone.

We have investigated the cellular and serum CK18 in 26 non-treated primary ductal invasive breast carcinomas. The soluble CK18 (TPS) was detected by chemiluminescent assay, and the cellular CK18 and PCNA expression by immunocytochemistry. Flow-cytometry was used to estimate the amount of DNA in malignant cells. There was a significant correlation between soluble CK18 and the pre-menopausal status (p0.05), characterized in our group by a PCNA estimated low proliferation index. We have also found a significant correlation between soluble CK18 and the DNA index (p0.01). The intracellular CK18 has correlated with the PCNA expression (p0.05), while no correlation could be found between cellular and serum CK18. The values of soluble CK18 may offer information about the treatment-induced cell death, if monitored, while isolated measurements should be interpreted cautiously. Elevated levels of serum CK18 in non-treated carcinomas may rather reflect a high tumor turn-over or perhaps a more intensive tumor cell killing.

In the last two decades, listeriosis, caused by the intracellular pathogen Listeria monocytogenes, became one of the most concerning food-born infections. Although it had been known before as an infectious disease of limited importance, listeriosis was brought into attention of scientists due to the frequent outbreaks recently reported. Despite the major progress made towards understanding the mechanisms of virulence of L. monocytogenes, our current knowledge into the process of Listeria-associated pathogenesis and virulence is still partial and fragmentary. In this study we demonstrate that T lymphocytes with reactivity to L. monocytogenes are frequently present in healthy individuals (73%), most probably as a consequence of subclinical infections. Host resistance to infection by L. monocytogenes involves a series of interactions between cells of the immune system, of which the antigen presenting cell/T lymphocyte partnership is essential. The ability of memory T cells to respond when exposed to their target antigen is traditionally assessed by measuring uptake of [3H] - thymidine. Our study has been carried out by means of an alternative methodology based on flow-cytometry, an approach which has several advantages on [3H] - thymidine incorporation technique: allows targeted analysis of particular cell types, simultaneous assessment of various cellular markers, and circumvents handling of radioisotopes.

The prediction of diabetes mellitus is mostly based on the existence of plasma markers. The aim of this preliminary study was to determine islet cell antibodies (ICA) and glutamic acid decarboxylase antibodies (GADA) in 28 diabetic children (12 of them having an evolutive disease of 1 year and 16 at the beginning of the diabetes) and to 47 of their first-degree relatives. There have been determined the levels of these two autoantibodies using the ELISA technique.To 17 of the patients with type I diabetes have been found high levels of GADA (60.7%) while 8 cases have positive ICA (28.5%). For the patients whose disease was diagnosed 1 year ago there have been found differences between the patients with and without antibodies regarding the level of the average values of Hb A1c, the daily insulin needs and the remission period. From the tested parents (a total of 25), 7 was GADA positive (28%), 6 had both antibodies present (24%) and one mother was ICA positive (4%). 9 of the brothers and sisters of the diabetic patients had high levels of GADA and 2 had both antibodies present. To the first-degree relatives with autoantibodies must be determined other plasma markers too (IAA, IA-2A) as well as genetic markers (HLA typing).The use of plasma markers is recommended as a first step in discovering the relatives with potential risk of developing the disease.

Both innate and adaptive immunity against Listeria monocytogenes are essential in circumventing or fighting the disease, although there is no clear delineation of the precise role of either in listeriosis. A consistent amount of in vivo experiments have generated a complex picture over the impact of listerial infection on the hosts, and they are shortly reviewed in this paper.

Since MM expands preferentially in the bone marrow (BM) and the close myeloma and stroma interaction is well recognized, we analyzed whether various soluble factors and different stroma can enhance in vitro MM growth. The expansion and protective function of various growth factors (A. 10ng/ml of each IL-6, VEGF, IGF, +/−super (s) IL-6; B. combination A. plus 10ng/ml of each VEGF, HGF, and IL13, and C. 10ng/ml IL-6, IGF, SDF1, Galectin and IL-1) and different stroma (BM stroma from healthy donors [BMSCs], a mouse stroma line [M210B4], and osteoclasts [OC]) were used for MM cell lines (CL: L363, U266, RPMI 8226). Cell number, viability, percentage and number of CD138+ cells were assessed at day 3 and 6 after culture. The most beneficial culture settings were used to establish their relevance for CD138+ enriched BM cells from MM patients (pts), propagated for up to 14 days in culture. MM cell growth proved to be induced through a cell contact mediated mechanism, this being cytokine and stroma dependent. The most beneficial cytokine combination for CL survival and proliferation was combination B, which could substitute the stroma support. Compared to the control, consisting of culture medium only (RPMI 1640+10%FCS), we detected a median 3.1- (combination B) vs. 1.9-fold increase (BMSCs) at day 3, and 1.24- vs. 1.2-fold increase in cell numbers at day 6. BMSCs proved to be most supportive, especially when used together with combination B (1.8- vs. 1.2-fold increase with BMSCs used alone on day 6 of culture), suggesting that BMSCs provide growth factors acting synergistically with IL-6+VEGF+IGF+sIL-6. The cell viability, albeit not cell expansion potential, was best preserved by OC. With proliferation of myeloma CL, a decrease in the percentage of CD138+ cells was observed, consistent with the reported phenotypic shift of plasma cells (PC) from CD138+ to CD138− during cytokine stimulation. Especially OC favoured the expansion of CD138- cells, suggesting that MM clones require defined conditions for their growth and survival. Culture conditions B and C, with or without BMSCs, could sustain primary BM cells from MM pts for up to 14 days. With the inherent variation of different MM pt samples, the combination of 5 factors (C) was more beneficial for these cells. An additional support for cell viability was provided by BMSCs, especially with longer culture periods (beyond 5 days). The different growth and survival requirements for CL seemed to also apply for CD138+ cells from MM pts, which demonstrated to be remarkably heterogeneous as determined by FACS analysis. We identified at least four different PC populations which coexisted before culture in the same specimen. The phenotype of these cells differed in terms of CD45, CD138, CD38, CD56, CD126, CD221, CD19 and CD28, both in their presence on the cell surface and intensity of expression. Taken together, our data indicate that various culture conditions show substantial differences in their ability to preserve myeloma cells, aimed to reproduce the BM microenvironment where the malignancy develops unconstrained. The availability of in vitro systems will allow testing of novel anti-MM agents, alongside with the development of human MM models in mice.