IgH基因重排和免疫分型检测在诊断B淋巴细胞增生性疾病中的初步应用

 目的 通过半巢式聚合酶链反应(PCR)技术对免疫球蛋白重链(IgH)基因重排的检测,建立对B淋巴细胞增生性疾病早期诊断的分子生物学方法,进而探讨在疑似B淋巴细胞增生性疾病诊断与鉴别诊断中的价值.方法 通过半巢式PCR技术检测8例已确诊的B淋巴细胞增生性疾病和24例临床疑似B淋巴细胞增生性疾病初诊患者的IgH基因重排;24例中流式细胞术检测了19例患者的免疫分型.结果 检测8例已确诊的B淋巴细胞增生性疾病,该方法敏感性为83.33%(5/6),特异性为100%(10/10);24例疑似B淋巴细胞增生性疾病初诊患者IgH基因重排检出的阳性率为83.33%(20/24),其中FR2A阳性率41.67%(10/24);FR3A阳性率50%(12/24);同时阳性2例.免疫分型结果14例可疑阳性,阳性率73.68%(14/19).结论 通过建立半巢式聚合酶链式反应(Polymerase chain reaction,PCR)技术检测IgH基因重排的分子生物学方法,对疑似B淋巴细胞增生性疾病患者的诊断起到了十分重要的作用,并且对反应性和肿瘤性淋巴细胞增生鉴别诊断具有重要的意义,为临床早期诊断、治疗干预提供了新的证据.     

治疗终止后儿童急性淋巴细胞白血病微小残留病的检测及临床意义探讨

以IgH和TCRγ两种基因重排为标志,通过多聚酶链反应(PCR)检测治疗终止后儿童急性淋巴细胞白血病(ALL)微小残留病(MRD).受检的18例病人中(化疗者11例,自体造血干细胞移植者7例),有9例于治疗终止后仍可检出MRD;其中的3例在检测后6个月内复发.治疗终止后未检出MRD的9例中无1例复发.化疗组与移植组MRD检出率无显著性差异(P=0.50)我们认为,治疗终止后仍可检出MRD者,其复发的危险性大,应定期监测体内MRD的变化.文中探讨了MRD检测的临床意义.本文应用的从存档骨髓涂片中提取NDA的方法具有简单、快速、可靠等优点.     

应用细胞周期阻断法分析初诊白血病患者骨髓及外周血微核率

目的：对初诊白血病患者骨髓及外周血进行微核分析。方法：应用细胞周期阻断法对54例初诊白血病患者骨髓及外周血与30例健康人外周血淋巴细胞进行微核检测。结果：54例初诊白血病患者骨髓及外周血淋巴细胞微核率（MNR）和微核细胞率（MCR）,与30例健康人外周血相比,P〈0.01。同一患者骨髓与外周血淋巴细胞MNR和MCR相比,P〉0.05。急性淋巴细胞白血病（ALL）、急性髓细胞白血病（AML）与慢性粒细胞白血病（CML）患者骨髓及外周血淋巴细胞MNR和MCR相比,P〈0.05。结论：白血病患者发病初期即存在染色体不稳定现象,不同类型白血病患者微核检测结果间有差异,推测与白血病的发病机制密切相关。     

CD40／CD40L在慢性淋巴细胞白血病治疗中的生物学意义

慢性淋巴细胞白血病（chronic lymphocytic leukemia．CLL）是一种主要源于B淋巴细胞的低度恶性小淋巴细胞疾病，其特征是成熟的小淋巴细胞在外周血、骨髓、淋巴结和脾脏中累积。大量研究表明．CLL细胞和其他B细胞恶性肿瘤一样．不能表达或弱表达共刺激分子，以致不能有效地递呈抗原及激发T细胞抗肿瘤的免疫反应，使得肿瘤细胞逃避免疫监视并不断增殖。     

高通量测序结合捕获技术检测非霍奇金B细胞淋巴瘤中重链基因重排VDJ区的临床研究

目的探讨高通量测序（Next-Generation Sequencing,NGS）结合基因捕获技术检测非霍奇金B细胞淋巴瘤中免疫球蛋白重链基因（Immunoglobulin heavy chain gene,IgH）重排的临床应用。方法收集2019年3月至2020年5月在中国人民解放军总医院第一医学中心治疗的15例成熟B细胞淋巴瘤病例,包括11例弥漫大B细胞淋巴瘤,1例滤泡性淋巴瘤,2例边缘区淋巴瘤,1例边缘区淋巴瘤伴局部弥漫大B转化淋巴瘤。采集其初诊时的组织样本与血浆样本,通过NGS检测IgH重排,并分析其与临床病理特征的相关性。结果在组织样本中,平均检出11 212条唯一的互补决定区3（Complementarity Determining Region 3,CDR3）序列,范围从515到41 476条;在血浆样本中,平均检出1 689条唯一的CDR3序列,范围从238到6 499条。在73.3%（11/15）的患者组织中检出特异性寡克隆扩增,在其对应的血浆样本中,72.7%（8/11）存在组织中的特异性寡克隆,并且二者的比例显著相关（Spearman rho=0.803,P=0.003）。另外,存在特异性寡克隆的组织样本中,克隆指数显著高于没有特异性寡克隆的样本（P<0.05）,表明存在特异性寡克隆的样本的IgH重排多样性较低。现有数据尚未发现克隆指数及组织中特异性寡克隆数与各项临床病理指标存在显著相关性。结论通过NGS检测非霍奇金B细胞淋巴患者的IgH重排,可以有效获得具体量化的CDR3序列,全面评估克隆多样性,并在大部分样本中得到特异性寡克隆序列。血浆检测结果与组织中的结果显著相关,可用于后续微小病灶残留的检测与复发监测。     

β-arrestin1和β-arrestin2在白血病中的表达及意义

目的探讨β-arrestin1和β-arrestin2在白血病患者中的表达及意义。方法收集初发白血病患者95例(Leu组),根据白血病类型不同分为:急性髓细胞白血病(AML)亚组(30例),急性淋巴细胞白血病(ALL)亚组(44例)和慢性粒细胞白血病(CML)亚组(21例);以非恶性血液病患者36例(NL组)作为正常对照。收集患者的骨髓和外周血标本,分离单个核细胞。采用实时定量PCR法检测β-arrestin1、β-arrestin2的mRNA表达,采用Western blotting和免疫荧光法检测β-arrestin1、β-arrestin2蛋白表达的变化。结果Leu组和NL组的骨髓和外周血单个核细胞中,均检测到β-arrestin1、β-arrestin2mRNA和蛋白的表达,Leu组显著高于NL组(P<0.01)。与NL组比较,AML、ALL和CML各亚组的骨髓和外周血中β-arrestin1、β-arrestin2的mRNA和蛋白表达水平均有明显升高(P<0.05或P<0.01),但三个亚组间无明显差异(P>0.05)。结论白血病患者骨髓和外周血单个核细胞中β-arrestin1、β-arresti...     

淋巴细胞指数在评估成人急性淋巴细胞白血病预后中的意义

目的探索成人急性淋巴细胞白血病(ALL)首次完全缓解(CR)时骨髓淋巴细胞指数检测及其在评估预后中的价值。方法回顾性分析近5年初治的经1～2个疗程诱导治疗达到完全缓解的205例ALL,按照首次CR时骨髓中原始细胞比例及淋巴细胞指数将其分为4组:A、B组骨髓原始细胞比例<1%,A组淋巴细胞指数<30,B组≤30;C、D组原始细胞比例≤1%,C组淋巴细胞指数>30,D组≤30。分析原始细胞比例?淋巴细胞指数与持续缓解时间(CCR)的关系。C、D组作MRD检测。结果A组原始细胞及淋巴细胞指数高于B组,CCR短于B组。C组与D组原始细胞比例相近,但C组淋巴细胞指数高,MRD高于D组,CCR时间短。C组CCR与B组无差异。结论CR后骨髓淋巴细胞指数与急性淋巴细胞白血病患者的CCR密切相关,它较原始细胞比例更准确?全面反映体内白血病细胞残留水平。淋巴细胞指数的检测有助于判断预后及指导今后的治疗。     

慢性淋巴细胞白血病遗传学异常及临床疗效和预后分析

目的:探讨应用荧光原位杂交技术(FISH)检测慢性淋巴细胞白血病(CLL)常见染色体异常,并分析其与临床治疗、预后的相关性。方法:对26例初诊CLL患者进行免疫表型及常规细胞遗传学(CC)检测,同时以4种序列特异性探针p53、D13S25、ATM、RB1和1种着丝粒探针CSP12,采用FISH对20例正常对照骨髓建立各探针的检测阈值,然后对26例CLL患者进行检测,检测结果大于阈值为阳性,小于阈值为阴性。结果:所有患者均表达CD19(100%),CD5阳性率为84.6%(22/26),CD23阳性率为65.4%(17/26),CD22阳性率为61.5%(16/26)。CC检测18.5%有核型异常(3/26),FISH检测出20例至少有1种基因异常,依次为D13S25缺失40.0%(8/20),RB1缺失25.0%(5/20),CSP12三体占15.0%(3/20),CSP12单体占5.0%(1/20),p53缺失10.0%(2/20),ATM缺失5.0%(1/20)。18例患者接受了含氟达拉滨的化疗(RF/F/FCR/FC),15例(83.3%)获完全缓解(CR)和部分缓解(PR),4例口服苯丁酸氮芥,4例接受CHOP方案联合化疗,1例获CR,2例PR。结论:FISH技术适用于CLL患者的细胞遗传学分析,提高了CLL遗传学异常检出率,FISH检测结果可为临床治疗方案选择提供一定的理论依据。氟达拉滨治疗方案能明显提高CLL患者长期生存率。     

γ-catenin在白血病细胞中的表达

目的检测白血病骨髓标本及白血病细胞株中γ-catenin的表达情况,探讨γ-catenin在白血病细胞中的作用。方法以RT-PCR检测76例白血病骨髓标本、8例正常骨髓标本和白血病细胞株K562、HL-60、U-937、Jurkat中γ-catenin的mRNA表达情况,以Western blot和免疫细胞化学法检测上述白血病细胞株中γ-catenin蛋白表达及其分布情况。结果正常骨髓标本中γ-catenin表达阳性率为0（0/8）,急性髓细胞白血病（AML）骨髓标本中γ-catenin表达阳性率为40.0%[12/30,其中M2型的阳性率为50.0%（7/14）],慢性髓细胞白血病（CML）骨髓标本中γ-catenin表达阳性率为13.6%（3/22）,急性淋巴细胞白血病（ALL）和慢性淋巴细胞白血病（CLL）骨髓标本中γ-catenin表达阳性率均为25.0%（3/12）。白血病组γ-catenin表达阳性率与正常组相比,AML组有显著性差异（P〈0.05,其中M2型最为显著）,其余各组均无显著性差异（P〉0.05）。RT-PCR及免疫组化检测可见HL-60和K562细胞γ-catenin呈强阳性,U-937和Jurkat细胞呈弱阳性表达,且γ-catenin主要表达于细胞质中。Western blot检测见K562和HL-60细胞γ-catenin呈阳性表达,而U-937和Jurkat细胞未见表达。结论作为细胞内信号传导分子,γ-catenin表达于AML组、HL-60及K562细胞中,可能与急性髓细胞白血病（特别是M2型）的发生有关。     

多聚酶链反应检测淋巴细胞白血病微小残留分析

根据IgH、TCR克隆性基因重排原理,用半重叠PCR和单轮PCR技术分别对同一份ALL患儿的骨髓标本进行检测(共25例),并对两法的检出率进行了比较。结果:半重叠PCR阳性捡出率为80％,单轮PCR阳性检出率为52％,前者较后者提高了28％,尤其对ALL-MRD组的检测,半重叠PCR的阳性检出率有显著提高。结果表明,半重叠PCR双轮扩增IgH、TCRγ基因重排对于MRD的检测起到增敏效果,可减少非特异性产物的生成,大大提高了MRD的阳性检出率,并与单轮PCR同样具有简便、快速以及用DNA量少,对DNA模板质量要求低,无放射性污染等优点。对区别肿瘤细胞的T、B细胞源性TCRβ基因重排尤其有价值。     

Chronic lymphocytic leukemia: Changes in bone marrow composition and distribution assessed with quantitative MRI

The purposes of this study were (α) to determine the prevalence of bone marrow abnormalities in patients with chronic lymphocytic leukemia (CLL) using quantitative MR assessment of axial marrow composition and peripheral marrow distribution; (b) to assess the agreement between both quantitative MR methods and compare their sensitivities to detect marrow alterations; and (c) to correlate MR findings with clinical and laboratory parameters. Twenty-nine consecutive patients with biopsy-proven CLL were investigated on a .5-T MR imager to determine bulk T1 relaxation times of the vertebral bone marrow and proportion of proximal femur surface area occupied by nonfatty marrow on coronal T1-weighted MR images of one hip. Of the 29 patients, 12 (41%) had abnormal increase in lumbar marrow T1 values (>600 msec) and 16 (55%) had increased proportion of surface area occupied by non-fatty marrow in the proximal femur (>+1 SD compared to normal values determined in sex- and age-matched healthy subjects). The results of both quantitative MR methods were normal in 12 patients and abnormal in 11 patients (agreement, 79%). Patients with alterations in peripheral marrow distribution had significantly higher T1 relaxation times (P = .001) than those with normal peripheral marrow. Patients with abnormal marrow composition or distribution at MRI had significantly higher blood and marrow lymphocytosis than patients without these features. In conclusion, the agreement between both quantitative MR methods suggests a parallelism between changes in axial marrow composition and in peripheral marrow distribution in patients with CLL. The limits of quantitative MRI in CLL must be kept in mind, because quantitative MR methods failed to detect leukemic marrow infiltration in 41% of patients.     

CT对慢性淋巴细胞白血病肝、脾体积与骨髓象、血象之间关系的研究

目的慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)的进展主要表现为大量淋巴细胞浸润至肝脏及脾脏内并导致肝脾肿大,本文采用IQQA-Liver自动分析软件测量肝、脾体积,探讨CLL肝、脾体积的变化是否同骨髓象、血象之间的变化存在相关性,为临床提供更多影像学信息及参考指标,辅助临床判断CLL患者病情的进展或缓解情况,同时亦为患者提供一个无创可靠的随访手段。方法回顾性收集15例经临床免疫表型分析、细胞流式术、骨髓活检等检查确诊(其中8例行个体化化疗)并复查随访的CLL病例,每次随访的患者前后均行全腹部或胸腹部MDCT扫描,且在行MDCT扫描的短期内(7天内)亦行骨髓象、血象等相关实验室检查,分析每例患者前后肝、脾体积变化与实验室骨髓象、血象变化之间的相关性。结果CLL患者的肝、脾体积及肝脾总体积与骨髓片、血片淋巴细胞比例计数之间有一定的相关性(r值分别为0.627、0.658、0.683及0.674、0.654、0.695,P值均<0.05)。结论MDCT辅以智能化自动分析软件可精确测量肝、脾体积,所测得的肝、脾体积的变化与骨髓、血片淋巴细胞计数变化呈中等程度相关,对CLL的进展、缓解及化疗效果的评估有一定的增益价值。     

Prolonged T1 relaxation of the hemopoietic bone marrow in patients with chronic leukemia

Eleven patients with chronic leukemia (7 with chronic lymphocytic leukemia and 4 with chronic myeloid leukemia) were evaluated with magnetic resonance (MR) imaging and T1 relaxation time measurements by use of a 1.5 tesla whole body MR scanner. Bone marrow biopsies were obtained from the posterior iliac crest (within 72 hours of the MR examination) in order to provide data on bone marrow cellularity and differential counts. The patients with chronic leukemia all showed a significant prolongation of the T1 relaxation times compared with the normal range for hemopoietic bone marrow.     

Initial experience with dynamic MR imaging in evaluation of normal bone marrow versus malignant bone marrow infiltrations in humans

The purpose of this study was (a) evaluation of dynamic contrast-enhanced MR imaging of normal bone marrow versus malignant bone marrow infiltrations in patients with proven B-cell-type chronic lymphocytic leukemia (B-CLL) and (b) correlation with the clinical stage according to Binet (stages A, B, C) and response to therapy. Bone marrow imaging of the lumbar spine, pelvis, and proximal femurs was performed at 1.5 T in 45 patients without known malignancy and in 30 patients with B-CLL. The differences between opposed-phase and in-phase dynamic gradient-echo sequences before and up to 10 minutes after intravenous application of .1 mmol/kg body weight of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) were evaluated in normal bone marrow. The contrast-enhancement patterns of normal and malignant bone marrow were compared using the opposed-phase dynamic gradient-echo sequence. Ten of the patients with bone marrow infiltrations (Binet stage C) additionally underwent MR imaging follow-up during therapy. Opposed-phase gradient echo sequences demonstrated a signal decrease of normal bone marrow, and in-phase gradient echo sequences demonstrated a signal increase of normal bone marrow after administration of Gd-DTPA. The dynamic signal intensity time courses differed significantly (P < .05) between Binet stages B and C and controls as well as among the three Binet stages of B-CLL. In the 10 patients followed during therapy, MR imaging sensitively demonstrated response (n = 6), nonresponse (n = 2), or relapse after initial response (n = 2). In out-of-phase imaging, both normal bone marrow and initial bone marrow infiltration in CLL stage Binet A show signal decrease after administration of contrast agent, whereas there is increase in signal intensity in higher-grade bone marrow infiltration in Binet stage B or C disease. The signal loss of normal bone marrow in out-of-phase imaging is a phase effect rather than a T2* effect. The differentiation of initial from higher-grade bone marrow infiltration on out-of-phase images relies solely on a shift in the fat/water ratio.     

Expression of the Bcl-2 family of proteins in peripheral blood B-lymphocytes in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD 5+ B cells in the early phase (G0/G1) of the cell cycle. It is considered that the accumulation of neoplastically transformed lymphocytes B (CLL cells) is primarily the consequence of the disturbance, i.e., blockade of these cells' apoptosis process. Apoptosis is the specific process of programmed cell death regulated by numerous extracellular and intracellular mechanisms. The Bcl-2 proteins are well-known modulators of this process. Some of these proteins (such as Bcl-2, and Bcl-XI) are anti-apoptotic, while others (such as Bad or Bax) are pro-apoptotic. Our study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens of healthy persons who represented the control group. Using Western blotting analysis, we quantitatively examined the protein expression of Bcl-2 family (Bcl-2, Bax, Bad, and Bcl-XI). The level of Bcl-2 (p = 3.68 x 10(-10)), Bax (p = 0.019), and Bad (p = 0.073) proteins expression was significantly increased in all the analyzed peripheral blood samples of patients, while the level of Bcl-XI protein (p = 0.75) did not significantly differ in peripheral blood samples of patients, compared to the controls. The results of this study showed that the increased level of expression of Bcl-2, Bax, and Bad protein represented the most striking feature of CLL cells. Moreover, the variations in the expression of only one protein of the Bcl-2 family could not represent the prognostic parameter in the treatment of this disease.     

Total-body irradiation with 25-MV photons in advanced non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Preliminary results and complications.

Patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma were treated with total-body irradiation (TBI). One group was treated after chemotherapy failed, while the other group received TBI initially. TBI was ineffective against CLL after chemotherapy failed. All patients with lymphocytic lymphoma who initially responded to chemotherapy but later relapsed were helped by TBI, as were 88% of patients with previously untreated lymphocytic lymphomas.     

Retrospective analysis of the clinical relevance of the Kiel classification of malignant non-Hodgkin's lymphomas (author's transl)]

405 cases with non-Hodgkin's lymphomas have been diagnosed according to the Kiel classification and analysed retrospectively. 314 patients with non-Hodgkin's lymphomas of low-grade-malignancy (chronic lymphocytic leukemia, lymphoplasmacytoid, centrocytic, centrocytic, centroblastic-centrocytic lymphoma) manifested significantly higher median survival times than the 91 patients with non-Hodgkin's lymphomas of high-grade malignancy (lymphoblastic and immunoblastic lymphoma). Within the group of patients with low-grade malignant lymphomas distinct prognostic differences were found whereas survival times in patients with lymphoblastic or immunoblastic lymphomas were rather similar. The lymphoblastic lymphoma showed a bimodal curve of age distribution whilst all other lymphomas had a maximum of incidence in the seventh decade of life. Increased frequency of B-symptoms did not necessarily represent an unfavorable prognostic factor for the lymphoma entity concerned. Except for chronic lymphocytic leukemia the highest incidence of initial bone marrow involvement was seen in lymphoplasmacytoid, centrocytic and lymphoblastic lymphomas. Centrocytes have been observed in peripheral blood of patients with centrocytic and centroblastic-centrocytic lymphomas, even though lymphocytosis did not exist. Monoclonal hypergammaglobulinemia was found in only 43% of the sera from patients with lymphoplasmocytoid lymphoma. In this disease, it was possible to differentiate between a lymphonodal, a splenomegalic and an extranodal manifestation.     

Detection of Richter's transformation of chronic lymphocytic leukemia by PET/CT.

Our objective was to evaluate the accuracy of PET/CT for the diagnosis of Richter's transformation of chronic lymphocytic leukemia (CLL) to diffuse large cell lymphoma. METHODS: A retrospective study was performed of 37 patients with CLL who underwent 18F-FDG PET/CT at our institution between March 2003 and July 2005. All PET/CT scans were reviewed in consensus by 2 diagnostic radiologists. Sites of abnormal 18F-FDG uptake with a maximum standardized uptake value (SUVmax) of greater than 5 were considered highly suggestive of Richter's transformation. The PET/CT findings were correlated with histologic findings from bone marrow or lymph node biopsy performed within 6 wk of PET/CT and with clinical follow-up. RESULTS: The 37 patients (26 men and 11 women; mean age, 61 y, range, 40-82 y) underwent 57 PET/CT scans. In 10 (91%) of 11 patients with Richter's transformation, PET/CT detected sites of abnormal 18F-FDG uptake having an SUVmax of greater than 5. Richter's transformation was missed in 1 patient who had only low-grade 18F-FDG uptake (SUVmax < 5). Nine patients had false-positive PET/CT findings; in 3 of these patients, alternative malignancies were diagnosed (Hodgkin's disease; metastatic neuroendocrine carcinoma; non-small cell lung cancer). In all remaining patients, PET/CT correctly excluded Richter's transformation. For the specific diagnosis of Richter's transformation of CLL to diffuse large B-cell lymphoma, PET/CT had overall sensitivity, specificity, and positive and negative predictive values of 91%, 80%, and 53% and 97%, respectively. CONCLUSION: PET/CT can detect Richter's transformation of CLL to diffuse large B-cell lymphoma with a high sensitivity and a high negative predictive value.