Spontaneous monoclonal immunoglobulin-secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M-Ig) production by circulating cells has not been reported. Using a two-colour ELISPOT assay, Ig-secreting cells (Ig-SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M-Ig in serum was greater than that of the other Ig-SCs. MM patients presented an excess of circulating heavy-chain (alpha or gamma) Ig-SCs (0.38% of the PBMC) with kappa or lambda light chains (0.48%) compared with the number of cells secreting the other heavy- (0.02%) and light-chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the mu-heavy-chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M-Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig-SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta-2 microglobulin concentration (r = 0. 69; P < 0.0003). The number of M-Ig-SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.     

Mobilisation of tumour cells along with CD34+ cells to peripheral blood in multiple myeloma

BACKGROUND: Cells belonging to the malignant clone are found in the peripheral blood in myeloma patients. In order to minimise the content of tumour cells in the stem cell product it is crucial to perform stem cell harvest at a time when tumour cells in the peripheral blood are at a minimum. OBJECTIVE: The aim of the study was to compare the mobilisation kinetics of normal CD34+ cells and myeloma plasma cells during mobilisation with either G-CSF alone or high-dose cyclophosphamide (HDCy) plus G-CSF. DESIGN AND METHODS: Morning blood samples were drawn each day during mobilisation from start of G-CSF or HDCy and to the end of leukapheresis, and were analysed by flow cytometry for content of CD34+ cells and myeloma plasma cells (CD38+ + CD45-). Tumour cells were also estimated by a patient-specific real-time polymerase chain reaction (PCR) method based on the 5' nuclease TaqMan technology. RESULTS: Flow cytometry data from 16 patients showed concomitant mobilisation of CD34+ cells and myeloma plasma cells. Seven patients were mobilised twice; first with G-CSF alone and then with HDCy plus G-CSF. There was no difference between the two mobilisation regimens regarding tumour cell mobilisation kinetics. Real-time PCR was performed in one patient and confirmed the mobilisation of tumour cells at the time when CD34+ blood cells were at a maximum. CONCLUSIONS: Tumour cells are mobilised to the peripheral blood at the same time as CD34+ cells in multiple myeloma patients after priming with both G-CSF alone and HDCy in combination with G-CSF.     

Detection of monoclonal plasma cells in the peripheral blood stem cell harvests of patients with multiple myeloma

We evaluated the harvest product from 47 patients undergoing peripheral blood (BP) stem cell collections for monoclonal plasma cells (PC) using a sensitive immunofluorescence technique. 60% (28/47) had docu mented tumour cells in the apheresis product. The 32 patients in plateau had a mean of 1.62 x 10⁶ PC/1 v 74.64 x 10⁶ in 15 relapsed patients ( P < 0.01). 32% (6/19) of patients without any tumour cells in the initial sample had them detected on a subsequent apheresis sample. In four cases (all treated with GM-CSF) small numbers of tumour cells were detected initially but became undetectable on a subsequent sample.     

Effect of interferon-α on immunoglobulin production by peripheral blood mononuclear cells in multiple myeloma

Abstract: To test a hypothesis that interferon-α (IFN) treatment might restore normal immunoglobulin (Ig) production in multiple myeloma (MM), the effect of IFN on Ig isotype (IgG and IgA) production by peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) in MM patients was analyzed by ELISA. IFN at a concentration of 1000 U/ml was found to enhance IgA production by PB MNCs in IgA-MM and had a trend to stimulate IgG production in IgG-MM. The effect of IFN on nonparaprotein Ig isotype production was more variable, with mostly neutral or inhibitory effects being seen in both the MM subtypes. In contrast to the influences observed in MM patients, IFN at the same concentration inhibited both IgG and IgA production by PB MNCs in healthy controls. In studying BM cells, IFN was found to reduce IgA production in IgA-MM, but had a neutral effect on IgG production in IgG-MM. In the controls, the production of both the IgG and the IgA isotypes by BM MNCs was decreased by IFN. On the basis of these results it seems that the disease itself somehow affects the Ig-producing cells in MM, when measured as different responses of the cells to exogenous IFN in vitro. The results do not support the hypothesis that IFN treatment could restore normal Ig production in MM patients.     

Detection of peripheral blood plasma cells as a predictor of disease course in patients with smouldering multiple myeloma

SUMMARY. We evaluated the number and labelling index of circulating peripheral blood monoclonal plasma cells (PBPC) in 57 patients with newly diagnosed smouldering multiple myeloma (SMM) to determine if these measurements could distinguish SMM from active multiple myeloma (MM). The PBPC were detected by a sensitive slide-based immunofluorescence procedure that identified PC by their morphology and monoclonal light chain staining. The presence of abnormal PBPC (defined as an increase in number or proliferative rate of circulating monoclonal plasma cells) was correlated with patient status at 6 and 12 months after testing. 16 of the 57 patients progressed within 12 months and 63% of these had abnormal PBPC. In contrast, only 10% (4/41) of those patients who remained stable had abnormal PBPC. The median time to progression for patients with abnormal PBPC was 0.75 years, compared to 2.5 years for those patients without abnormal PBPC ( P < 0.01). The detection of PBPC is important and helps to identify patients with active MM when other parameters suggest SMM. The lack of abnormal PBPC suggests SMM, and these patients may have a stable course without the need for immediate chemotherapy.     

多发性骨髓瘤患者外周血单个核细胞中糖皮质激素诱导的亮氨酸拉链蛋白mRNA表达的研究

目的检测多发性骨髓瘤（MM）患者外周血单个核细胞中糖皮质激素诱导的亮氨酸拉链蛋白（GILZ）mRNA表达情况,探讨GILZ在多发性骨髓瘤骨病治疗中的作用。方法选择使用VAD方案治疗后的MM患者（研究组）、初次发病的MM患者（病例对照组）及健康体检者（正常对照组）各11例作为研究对象。采用RT—PCR法检测外周血单个核细胞中GILZmRNA的表达,采用ELISA法检测血清骨碱性磷酸酶（BALP）和骨钙素（OT）的表达。结果与研究组相比,病例对照组GILZmRNA、BALP和OT表达水平均明显降低,且差异有统计学意义;正常对照组GILZmRNA、OT表达水平降低,BALP表达水平升高,且差异均有统计学意义。病例对照组GILZ表达与BALP、OT表达均呈正相关性;研究组GILZ表达与BALP、OT表达也均呈正相关性。结论地塞米松可以明显上调MM患者GILZmRNA表达;GILZmRNA表达上调可以促进成骨标志物BALP、OT表达,缓解MM患者骨质的破坏。     

Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors

Using a combination of GM-CSF, SCF, flk-2/flt-3 ligand, and IL-4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10 d of culture were 8.19×10⁵ and 9.87×10⁵ cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA-DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was considered a prerequisite for future investigations directed towards developing effective immunotherapies for myeloma.     

结核潜伏感染者全血miR-144-3p、miR-146a-5p的表达

目的探讨结核潜伏感染者全血miR-144-3p、miR-146a-5p的表达及其与活动性肺结核患者的差异。方法收集结核潜伏感染者30例,活动性肺结核患者30例。收集全血,提取总RNA,利用反转录-荧光定量PCR方法检测miR-144-3p、miR-146a-5p的表达。两组间比较采用t检验。结果结核潜伏感染者全血中miRNA144-3p的表达(2.014±1.48)比活动性肺结核患者(1.056±0.746)明显提高,差异有统计学意义(P<0.05),而结核潜伏感染者全血中miR-146a-5p的表达(1.937±1.109)与活动性肺结核患者(1.469±0.693)相似,差异无统计学意义(P>0.05)。结论 miR-144-3p可能成为诊断结核潜伏感染者的标志物。     

High numbers of clonal CD19+ cells in the peripheral blood of a patient with multiple myeloma

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 (<1%) post transplant, even after disease progression. In addition, we found that the MM clone did not include a B-progenitor population defined by CD34.     

冠心病患者外周血单核细胞中miR-125a-3p的表达研究

目的 探讨冠状动脉粥样硬化性心脏病(coronary heart disease,CHD)患者外周血单核细胞中miR-125a-3p的表达水平.方法 收集昆明医科大学附属延安医院心血管内科2014年8月1日至2014年10月30日收治并被确诊为冠心病的患者共63例,其中稳定型心绞痛组23例(B组);急性冠状动脉综合征组40例(C组),选择健康对照组20例(A组).抽取静脉血,分离单核细胞,提取单核细胞总核糖核酸(ribonucleic acids,RNA),使用实时荧光定量PCR(real-time fluoresecnt quantitative PCR,RT-PCR)检测miR-125a-3p的表达水平.结果 A组、B组、C组miR-125a-3p表达水平分别为1、3.12(2.86~3.38)、24.79(8.85~40.73).与A组比较,B组、C组miR-125a-3p表达水平显著上调(P<0.01);与B组比较,C组miR-125a-3p表达水平显著上调(P<0.01).结论 miR-125a-3p可能参与动脉粥样硬化的发生和进展,可能为动脉粥样硬化治疗的新靶点.     

Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK-associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56⁺ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16⁺ cells did not differ significantly from that of normal BM samples. The number of CD56⁺CD3^? lymphocytes increased significantly within high-risk patients (869 ± 671) as compared to intermediate (388 ± 212) and low-risk patients (274 ± 199) (P = 0.04). Moreover, patients with high values of CD56⁺CD3^? lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high β2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56⁺CD3^? lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57⁺CD8^? cells had a better outcome.     

The relationship between monoclonal myeloma precursor B cells in the peripheral blood stem cell harvests and the clinical response of multiple myeloma patients

The aim of this study was to determine the presence of monoclonal myeloma precursor B cells in peripheral blood stem cell harvests and to investigate their role in the clinical outcome of multiple myeloma patients. A total of 39 multiple myeloma patients were treated with a sequential therapy including double high-dose melphalan therapy followed by a double transplant procedure. The apheresis products for the second transplant were purged using a panel of four or five different mouse monoclonal antibodies against B-cell antigens (CD10, CD19, CD20, CD22 and CD37). In 19/39 patients a tumour-specific CDR III signal was identified in the diagnostic bone marrow. Gene scan analysis after CDR III PCR of the magnetic bead isolated B-cell fraction from the apheresis products in these 19 patients revealed three different patterns: 32% of patients had a predominantly monoclonal B-cell population; 63% of patients had an identifiable monoclonal signal within an oligoclonal B-cell population. In only 1/19 patients were no monoclonal B cells identified in the B-cell population of the apheresis product. A correlation between the clonal pattern and the clinical response after sequential chemotherapy was found. Patients with a predominance of monoclonal myeloma or myeloma precursor B cells had an early relapse or achieved a minimal response or a partial remission. Patients with an oligo- and/or polyclonal pattern achieved a high percentage of partial as well as complete remissions.     

Factors influencing collection of peripheral blood progenitor cells following high-dose cyclophosphamide and granulocyte colony-stimulating factor in patients with multiple myeloma

We treated 103 multiple myeloma (MM) patients with 7 g/m² cyclophosphamide (Cy) followed by 300 μg G-CSF/d to harvest peripheral blood progenitor cells (PBPC). PBPC autografts containing > 2.0 × 10⁶ CD34⁺ cells per kg body weight were obtained at the first attempt from 90/100 evaluable patients. The most significant factor predicting impairment of PBPC collection was the duration of previous melphalan treatment ( P  < 0.0001). In multivariate discriminate analysis, treatment with melphalan during the most recent chemotherapy cycles prior to mobilization ( P  = 0.0727) and previous radiotherapy ( P  = 0.0628) had a marginally significant negative influence on the efficacy of PBPC collection. We found no reduced functional capacity of CD34⁺ cells to restore haemopoiesis after myeloablative treatment related to the duration of melphalan exposure. At the time of best response to conventional treatment, a median paraprotein reduction of 21% was achieved following high-dose cyclophosphamide (HD-Cy). Two heavily pretreated patients died and one patient developed pulmonary toxicity W.H.O. grade IV following HD-Cy. Potential transplant candidates should undergo mobilization and harvesting of PBPC before melphalan-containing treatment. Combinations of haemopoietic growth factors and their dose modifications should be investigated to improve PBPC collection, to allow a dosage reduction of the mobilization chemotherapy.     

Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti-idiotype vaccination

Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti-idiotype vaccination were labelled with clinical-grade anti-CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal-calf-serum-free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte-macrophage colony stimulating factor plus interleukin 4 (IL-4), followed by either tumour necrosis factor-alpha (TNF-alpha) or a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. The CD14+ cell yield was increased from 17.6 +/- 6.5% to 93.8 +/- 6.3% (recovery 64.4 +/- 15.4%, viability > 97%). After cell culture, phenotypic analysis showed that 86.7 +/- 6.8% of the cells were DC: 2.27 +/- 0.9 x 108 DC/leukapheresis were obtained, which represented 20.7 +/- 4.6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28.6 +/- 3% of initial CD14+ cells) of DC than TNF-alpha alone, with secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 +/- 10%. Finally, interferon-alpha-2b was at least as efficient as IL-4 in inducing the differentiation of mature, functional DC from monocytes.     

Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.     

Deficiency of Con A-induced suppressor cell activity in peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax

Con A-pretreated mononuclear (MNC) cells from Thai adults with naturally acquired P. falciparum or P. vivax malaria were significantly less effective in suppressing the responsiveness of autologous or normal allogeneic responder cells to mitogenic lectins or allogenic stimulator cells than pretreated cells from healthy donors. Serial studies of three patients demonstrated that reduced suppressor cell activity was present early in malaria infection but returned to normal soon after treatment. These studies demonstrate that the loss of T cells previously observed in patients with malaria, in part may functionally represent a loss of suppressor T cells.     

Feasibility and toxicity of high-dose therapy (HDT) supported by peripheral blood stem cells in elderly patients with multiple myeloma and non-Hodgkin's lymphoma: survey from a single institution

The aim of this retrospective study was to investigate the feasibility of high-dose therapy (HDT) followed by peripheral blood stem cell transplantation (PBSCT) in elderly patients with hematological malignancies. From April 1998 to November 2001, 40 elderly patients (defined as > or =60 years) with non-Hodgkin's lymphoma (12 patients) and multiple myeloma (28 patients) were evaluated. Seven lymphoma and one myeloma patients were in complete remission (CR), 27 in partial remission (PR), two had stable disease (SD), and three progressive disease (PD). The median age was 65 years (range 60-71). Thirty-nine patients were mobilized with chemotherapy plus granulocyte-colony stimulating factor (G-CSF) and one with G-CSF alone. Patients received HDT including melphalan alone in 32 cases or combined with other drugs in six and BEAM in two. The median number of collected CD34(+) cells was 12.4 x 10(6)/kg (range 2.0-68.9). The median number of re-infused CD34(+) cells was 9.9 x 10(6)/kg (range 2.0-68.9). All patients engrafted after PBSC and the median time to neutrophil recovery (N > 500/micro l) and platelet recovery (PLT > 20,000/micro l) was 8 days (range 5-18) and 6 days (range 5-18), respectively. Nonhematological toxicity was mild and no patient died from transplant-related toxicity (TRM). Median duration of hospitalization was 18 days (range 12-24). To date, 32 patients are alive and eight died from disease progression at a median follow-up interval of 24 months. HDT supported by PBSC is a feasible procedure in selected elderly patients, and an age of more than 60 years should not be considered a contraindication for HDT.     

Interleukin-1 in multiple myeloma: producer cells and their role in the control of IL-6 production

We studied the role of interleukin (IL)-1β in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL-1β gene and produced IL-1β. Myeloma cells less potently expressed the IL-1β gene and IL-1β protein. IL-1β gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL-1β gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL-1 was mainly responsible for IL-6 production in the tumoural environment through a PGE₂ loop. In fact, an IL-1 receptor antagonist (IL-1RA) blocked PGE₂ synthesis and IL-6 production by 80%; this blockage could be reversed by adding synthetic PGE₂. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE₂ synthesis. Taken together, these data emphasize the possibility of blocking IL-1 by using IL-1RA or other antagonists in order to block IL-6 production, which is a major tumoural survival and proliferation factor.     

Long-term effects of idiotype vaccination on the specific T-cell response in peripheral blood and bone marrow of multiple myeloma patients

OBJECTIVES: To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). MATERIALS AND METHODS: Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (gamma-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. RESULTS: At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th(2) cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) (P < 0.02) and shifted at the gene level from a Th(1) to a Th(2) profile (P < 0.05). CONCLUSION: Id-specific T-cells may decline overtime and shift toward a Th(2) response and may be found at a similar frequency of patients in blood and BM.