石蜡包埋B细胞性恶性淋巴瘤组织克隆性免疫球蛋白重链基因重排检测研究

[目的]探讨克隆性免疫球蛋白重链(IgH)基因重排在B细胞性非霍奇金淋巴瘤(B-NHL)诊断中的价值.[方法]采用半巢式PCR方法,对81例B-NHL病例,36例反应性增生及12例非淋巴瘤组织样本进行克隆性免疫球蛋白重链基因重排检测,以上病例均为经福尔马林固定的石蜡包埋组织.[结果]81例B-NHL中68例IgH阳性(阳性率为83.95%),36例反应性增生均为多克隆性,12例非淋巴瘤组织样本结果IgH均为阴性.[结论]克隆性免疫球蛋白重链基因重排检测可以作为诊断恶性B细胞性非霍奇金淋巴瘤的有效分子标志.     

自体外周血造血干细胞移植治疗多发性骨髓瘤16例临床疗效观察

目的:评价自体外周血造血干细胞移植(autologousperipheralbloodstemcelltransplantation,APBSCT)治疗多发性骨髓瘤的临床疗效。方法:16例确诊多发性骨髓瘤患者接受APBSCT,其中2例接受了二次移植,1例接受CD34+细胞筛选后的自体外周血造血干细胞移植。移植后继续常规化疗,13例患者给予α-干扰素维持治疗。结果:APBSCT可延长多发性骨髓瘤患者的无瘤生存率及总生存率,该组患者3年、5年无瘤生存率分别为18.75%±9.75%、0,平均无瘤生存时间为24.8个月。3年、5年总生存率分别为41.25%±12.72%、18.33%±10.77%,平均总生存时间为37.4个月。本组移植患者的CR率高,达76.92%,接近国外报道。而且,移植后造血重建快,移植相关并发症少。结论:APBSCT是治疗多发性骨髓瘤、改善其预后的重要手段。     

Distinctive Features of Immunoglobulin Heavy Chain Variable Region Gene Rearrangement in Multiple Myeloma

We have analysed the rearranged Ig heavy chain (IgH) genes in a series of 28 cases of multiple myeloma (MM), in order to extend the study of Ig heavy chain variable (V_H) gene usage in B lymphoid malignancies and to explore the ontogenic compartment from which transformed precursor cells arise in this disease. We were able to amplify 28 rearranged alleles by polymerase chain reaction from 23 of these cases, using a common joining region (J_H) amplimer together with a panel of V_H family-specific amplimers. The pattern of VH family usage was similar to that reported in normal peripheral blood B cells with infrequent usage of V_H5 and V_H6 genes. However, nucleotide sequence analysis of 17 IgH alleles revealed rearrangement of other V_H family members, closely related to known developmentally regulated V_H genes, some of which are known to be associated with autoimmune specificities. In contrast to previous findings on more immature B lineage malignancies, the rearranged genes diverged extensively from consensus germline sequences, consistent with somatic mutation. These findings support the hypothesis that the major proliferating precursor in MM arises at, or following a stage of T cell-dependent germinal centre proliferation in lymphoid follicles.     

Prognostic Significance of Immunoglobulin and T Cell Receptor Gene Rearrangements in Patients with Acute Myeloid Leukemia: Taiwan Experience

We investigated the prognostic significance of lymphoid antigen receptor gene rearrangement in patients with newly diagnosed acute myeloid leukemia (AML). Thirty-nine patients were included in the study. Clonal gene rearrangement of immunoglobulin heavy chain (IgH) and T cell receptor β chain (TCRβ) was found in leukemic cells in 11 (28.2%) and 10 (25.6%) patients, respectively. Five (12.8%) had both IgH and TCRβ gene rearrangements. Three of the seven (42.9%) B-lymphoid marker-positive and eight of the 32 (25%) B-lymphoid marker-negative patients had clonal IgH gene rearrangements. Five of the 11 (45.5%) T-lym-phoid marker-positive and 5 of the 28 (17.9%) T-lymphoid marker-negative patients had clonal TCRβ gene rearrangements. All patients were treated with similar regimens. The complete remission rate (62.5% vs 65.296, p=1.000) and median survival (13 vs 14 months, p=4.366) were similar in patients with and without clonal IgH or TCRβ gene rearrangements. In conclusion, while clonal rearrangements of IgH or TCRβ genes were found in AML patients, they did not appear to effect the prognosis.     

Deep Sequencing Reveals Myeloma Cells in Peripheral Blood in Majority of Multiple Myeloma Patients

IntroductionThe evaluation of myeloma cells in multiple myeloma (MM) patients has generally been limited to the assessment of bone marrow involvement because of the sensitivity limitations of traditional minimal-residual-disease–detection methods.Materials and MethodsWe developed a sequencing-based method to identify myeloma cells in bone marrow (BM) and peripheral blood (PB) samples, based on their unique immunoglobulin gene rearrangements, that can detect cancer clones at levels well below 1 in 1 million leukocytes (0.0001%). In this multisite study, we used this sequencing method to determine the fraction of patients with myeloma cells in their PB at diagnosis and posttreatment time points.ResultsUsing this sequencing approach, we detected myeloma cells in the PB in the vast majority of MM patients (44/46, 96%). We demonstrated a clear correlation (R² = 0.57) between myeloma clone levels in paired BM and PB samples, and noted that PB clone levels were approximately 100-fold lower than levels in BM samples. The sequencing assay demonstrated a clear sensitivity advantage in the BM compartment and at least equivalent sensitivity in the PB compared with that of monoclonal-protein results.ConclusionThis study highlights the promise of a blood-based, sequencing minimal-residual-disease assay that can be used to measure MM disease burden at different time points and various disease stages.     

Reduction of Allogeneic Transplant Morbidity by Combining Peripheral Blood and Bone Marrow Progenitor Cells

One case has been reported of a patient undergoing allogeneic transplantation with peripheral blood progenitor cells (PBPCs) rather than bone marrow.¹ We now report the first case of a patient who underwent an allogeneic transplant with bone marrow combined with PBPCs.     

慢性淋巴细胞白血病伴多发性骨髓瘤:共同克隆起源的证明

研究一例慢性 B 淋巴细胞白血病(CLL)伴多发性骨髓瘤(MM)的患者。用 FACS 预先分离 CLL 和 MM 的细胞。免疫球蛋白的重链基因的免疫球蛋白的决定互补区Ⅲ(CDRⅢ)DNA 序列分析显示在 CLL 和 MM 细胞群有相同的基因重排。本研究证明 CLL和 MM 均有共同的克隆起源。     

慢性淋巴细胞白血病伴多发性骨髓瘤:共同克隆起源的证明

研究一例慢性B淋巴细胞白血病(CLL)伴多发性骨髓瘤(MM)的患者。用FACS预先分离CLL和MM的细胞。免疫球蛋白的重链基因的免疫球蛋白的决定互补区Ⅲ(CDRm)DNA序列分析显示在CLL和MM细胞群有相同的基因重排。本研究证明CLL和MM均有共同的克隆起源。     

The Role of Stem Cell Factor in Mobilization of Peripheral Blood Progenitor Cells

Stem cell factor (SCF) is a hematopoietic growth factor which acts on both primitive and mature progenitors cells. In animals, high doses of SCF alone stimulate increases in cells of multiple lineages and mobilize peripheral blood progenitor cells (PBPC). Phase I studies of rhSCF have demonstrated dose related side effects which are consistent with mast cell activation. Based upon in vitro synergy between SCF and G-CSF we have demonstrated the potential of low doses of SCF to synergize with G-CSF to give enhanced mobilization of PBPC. These PBPC have increased potential for both short and long term engraftment in lethally irradiated mice and lead to more rapid recovery of platelets. On going Phase I/II studies with rhSCF plus rhG-CSF for mobilization of PBPC, demonstrated similar increases in PBPC compared to rhG-CSF alone. These data suggest a clinical role of rhSCF in combination with rhG-CSF for optimal mobilization of PBPC.     

Ultrastructural Features of CD34+ Hematopoietic Progenitor Cells from Bone Marrow, Peripheral Blood and Umbilical Cord Blood

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomag-netically separated CD34⁺ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34⁺ cells from PBSC were morphologically very homogeneous and 94.7 ± 4.5% of these cells were MPO^-: these ultrastructural features are generally considered typical of immature cells. The CD34⁺ BM cells were instead more heterogeneous, with 24.6 ± 7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.     

Molecular Diagnosis of Micrometastasis in Lymph Nodes,Peripheral Blood and Bone Marrow in patients

Background and Objective Lymph node, peripheral blood and bone marrow from NSCLC patients have undetectable micro-metastasis by general method, and the tumor micrometastasis is     

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.     

Plasma cell tumors in HIV-positive patients: report of a case and review of the literature

Plasma cell tumors show an increased incidence in HIV-positive patients. The cases reported in the literature suggest that plasma cell tumors occur in a younger age group than that encountered in the general population. Pathologically, many of these tumors show a plasmablastic morphology. Plasma cell tumors in HIV-positive patients may present at unusual sites and progress rapidly to involve multiple sites, including the soft tissues and viscera. The prognosis is generally poor. These features may be related to a combination of factors, including immunodeficiency, oncogenic viruses, and altered cytokine milieu in these patients. A case of plasma cell tumor in an HIV-positive patient is presented.     

外周血细胞计数及其比值对初诊伴非骨相关髓外病变的多发性骨髓瘤患者预后的评估价值

 目的 探讨外周血淋巴细胞计数（ALC）、单核细胞计数（AMC）、淋巴细胞与单核细胞比值（LMR）、血小板与淋巴细胞比值（PLR）与初诊时伴非骨相关髓外病变（sEMD）的多发性骨髓瘤（MM）患者临床病理特征的相关性以及对疗效和生存的影响。方法 收集81例初诊时伴sEMD的MM患者临床病理资料，分别分析外周血ALC、AMC、LMR、PLR与血红蛋白、肌酐水平、乳酸脱氢酶水平、β2微球蛋白水平、治疗疗效、预后生存情况等指标关系。以ALC、AMC、PLR、LMR中位数为界值进行分组。Kaplan-Meier法分析ALC、AMC、PLR、LMR与生存及预后之间的关系；预后多因素分析采用Cox风险回归模型。结果 81例患者ALC、AMC、PLR、LMR中位数分别为1.38×10⁹/L、0.48×10⁹/L、134.9、3.11，多因素分析结果显示：LMR≤3.11（P=0.021）、PLR≥134.9（P=0.019）、LDH≥247U/L（P=0.041）、Hb≤110 g/L（P=0.004）是初诊伴sEMD的MM患者预后不良的影响因素。结论 对于初诊伴sEMD的MM患者，LMR≤3.11、PLR≥134.9、LDH≥247 U/L、Hb≤110 g/L可能是其影响预后不良的独立因素。     

Clinical Significance of bcr-abl Gene Rearrangement Detected by the Polymerase Chain Reaction After Allogeneic Bone Marrow Transplantation in Chronic Myelogenous Leukemia

Although serial detection of bcr-abl positive cells by PCR appears able to identify distinct patient groups with different risks of relapse following BMT, there remain many unanswered questions regarding the clinical utility and biological significance of PCR detectable cells in this disease. Many of the studies summarized have conflicting results and the influence of various clinical parameters which are known to affect the risk of relapse post-BMT has not yet been consistently associated with the ability to detect bcr-abl positive cells by PCR. These clinical parameters include GVHD, T-cell depletion and intensity of immunosuppression following BMT. Prospective studies with larger patient numbers will be necessary to define the impact of these factors in PCR status and relapse. The answers to all these questions will increase our understanding of the biology of chronic myelogenous leukemia and help provide more effective therapies for the future.     

Prognostic Significance of Bone Marrow Trephine and Peripheral Blood Smears in 55 Patients with Mantle Cell Lymphoma

Bone marrow trephine and peripheral blood smears taken at diagnosis of 55 cases of well-documented mantle cell lymphomas were reviewed in order to analyse the leukaemic involvement in this non-Hodgkin's lymphoma: its incidence, morphological characteristics and prognostic significance. A median survival of 36 months was found. The median age was 61 and the male to female ratio was 4:1. Morphologically 7 cases presented with a mantle zone pattern, all the others had a diffuse pattern. Involvement of the bone marrow was found in 58% and a trend for prolonged survival in patients with a negative trephine was seen. An absolute lymphocytosis above 10,000 μ1 was found at diagnosis in 5 cases (10%) and had a statistically significant impact on survival. An additional 5 cases developed frank leukaemia during the course of the disease and died within 1 to 6 months of this evolution, suggesting that marked lymphocytosis is more a terminal event associated with an extremely poor prognosis than a presenting symptom. Finally we identified an additional parameter with statistically prognostic significance, namely, the presence of atypical cells in the peripheral blood even in the absence of an increased lymphocytosis.     

异基因外周血干细胞移植后巨细胞病毒感染的早期检测及临床意义

目的:选择有效的巨细胞病毒(CMV)早期检测方法用于异基因外周血干细胞移植(allo-PBSCT).方法:采用微粒子酶免疫分析法(MEIA)、间接免疫荧光法(IFA)及实时荧光定量PCR法(Real-time FQ-PCR)对42例allo-PBSCT患者移植后不同时间进行多点动态检测外周血 CMV IgM 抗体、CMV pp65 抗原及 CMV DNA 负荷量.结果:1)移植患者CMV pp65抗原及DNA负荷量的阳性检出率(38.24%,33.82%)明显比IgM抗体阳性检出率(21.50%)高(P＜0.05).2)CMV pp65抗原及DNA负荷量的阳性检出时间(75±64天,60±50天)明显早于IgM(127±73天)(P＜0.05).3)CMV活动性感染的发生与患者移植后急性移植物抗宿主病(aGVHD)的发生密切相关(P＜0.05).结论:pp65抗原和DNA负荷量的检测方法适合用于allo-PBSCT术后患者的CMV感染的早期诊断.aGVHD的发生是allo-PBSCT患者CMV活动性感染的危险因素.     

急性白血病异基因外周血造血干细胞移植血缘供者安全性分析

目的：探讨急性白血病异基因外周血造血干细胞移植血缘供者近期的不良事件及远期的安全性。方法185例急性白血病异基因外周血造血干细胞移植血缘供者,干细胞动员后,用COBE Spectra血细胞分离机采集外周血干细胞。检测供者动员时血常规及形态学分类、CD34＋细胞百分比、干细胞混悬液中单个核细胞（ MNC）数量及CD34＋细胞计数,同时记录供者动员和采集过程中的不良事件、采集后第1、3、7天血常规变化及长期随访。结果血缘供者干细胞动员前白细胞计数为（6.16±1.58）x 109·L-1,采集时白细胞计数为（41.55±9.77）x109·L-1,是动员前6.75（4.18-10.87）倍。干细胞混悬液MNC为（247.61±105.99）x 108,CD34＋细胞为（3.54±3.42）x 108。动员时血缘供者的常见不良事件有头痛、腰背困痛、四肢痛、全身痛,合计占94.6%;外周血干细胞采集过程中,静脉持续输注葡萄糖酸钙,供者口周和指尖麻木等低钙症状发生率为3.2%;所有供者的不良事件均可耐受,无一例供者因不良事件终止干细胞动员或采集。供者干细胞采集后1周血象基本恢复正常;随访11 a血缘供者健康状况良好,无与动员及采集相关的远期并发症发生。结论急性白血病异基因外周血造血干细胞移植血缘供者动员采集无明显的近期不良事件及远期并发症,安全性非常高,为非血缘移植供者提供可靠的安全保障依据,减少恐惧感,降低反悔率。     

多周期大剂量化疗辅以外周血干细胞支持对难治性卵巢癌的治疗探索

卵巢癌的复发和耐药是治疗的重点和难点。目前 ,仍没有较为可靠的治疗手段 ,多周期、大剂量化疗辅以外周血干细胞回输支持 ,作为复发或难治性卵巢癌的巩固或一线治疗方案 ,则能够明显减少大剂量化疗引起的毒副反应 ,缩短化疗周期 ,国外已进行了多中心、多周期、多方案的临床探索 ,有望成为治疗此类卵巢癌的有效方案。