单克隆IgH基因重排检测在B-NHL诊断和随访中的意义

目的　评价单克隆IgH基因重排检测在恶性淋巴瘤 (B NHL)临床中的应用价值。方法　用半巢式PCR检测单克隆IgH基因重排。病例组为B NHL ,包括 6 9例石蜡包埋组织切片、治疗前 16例骨髓和 2 9例外周血、阳性者治疗后复查骨髓和外周血 ;对照组为 10例慢性淋巴结炎、3例T NHL和 2例HD。结果　对照组均阴性。病例组 :切片中单克隆IgH基因重排阳性率为 6 3.8% (44 / 6 9) ;骨髓和外周血阳性率分别为 43 .8%(7/ 16 )和 41.4% (12 / 2 9) ,细胞形态学检查未见异常细胞者阳性率分别为 33 .3% (3/ 9)和 31.3% (5 / 16 )。 16例同时采集骨髓和外周血者 ,阳性率分别为 43 .8% (7/ 16 )和 37.5 % (6 / 16 ) ,两者无统计学差异。治疗前单克隆IgH重排阳性者 ,6例完全缓解 (CR)后转阴 ,处于持续缓解状态 ,1例临床缓解后 13个月仍阳性 ,现在继续随访中 ,另 1例CR后持续阳性者 ,6个月后复发。结论　切片、骨髓和外周血中检测单克隆IgH基因重排可以作为B NHL诊断和随访微小残留病灶的辅助手段     

Molecular monitoring of tumour load kinetics predicts disease progression after non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

BACKGROUND: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting. PATIENTS AND METHODS: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested. RESULTS: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/10(4)-1/10(5) cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse. CONCLUSION: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.     

实时定量PCR检测B细胞非霍奇金淋巴瘤患者外周血和骨髓IgH基因重排及临床意义

目的:检测B细胞非霍奇金淋巴瘤(B-NHL)患者外周血和骨髓中IgH基因重排并探讨其在临床诊治中的应用。方法:利用SYBR GreenⅠ荧光染料,采用实时定量PCR方法,以IgH基因为标志,对B-NHL患者治疗后采集的15例外周血及10例骨髓的IgH基因重排进行定量分析。结果:15例外周血及10例骨髓均检测到IgH基因拷贝数,外周血和骨髓差异无统计学意义(P>0.05)。结论:实时定量PCR方法对外周血和骨髓中IgH基因重排定量分析,可以作为B-NHL鉴别诊断和随访微小残留病的辅助手段,并对判断疗效、预测复发有一定的临床意义。外周血和骨髓IgH基因拷贝数差异无统计学意义。     

实时定量PCR法检测淋巴瘤患者Bcl-2／IgH融合基因及其临床意义

本研究目的是检测滤泡性淋巴瘤和弥漫大B细胞淋巴瘤患者bcl-2／IgH融合基因的表达水平,以探讨其临床意义。以SYBRGreenI荧光染料实时定量PCR方法检测20例患者外周血和（或）骨髓bcl-2／IgH融合基因的表达水平,并对其中4例患者bcl。2／IgH融合基因的表达水平进行动态监测。结果表明,在bcl-2／IgH融合基因阳性表达的病例中,骨髓和外周血bcl-2／IgH融合基因的相对拷贝数分别为4．23和2．73,统计学分析差异无显著性（P=0．107）,而治疗前后融合基因的相对拷贝数均数分别为3．61和2．69,统计学分析差异有显著性（P=0．000）,初诊及复发组的bcl-2／IgH融合基因表达水平明显高于缓解组（P=0．008）,在4例患者的外周血bcl-2／IgH融合基因动态监测结果中,l例患者在临床复发前3个月bcl-2／IgH融合基因表达水平明显上升。结论：bcl-2／IgH融合基因表达水平与患者的疾病状态有一定的相关性,初诊及复发患者融合基因的表达水平高,而缓解患者融合基因的表达水平低,治疗后融合基因表达水平较治疗前明显下降,bcl／IgH融合基因表达水平的变化可能与临床疾病进程有关,检测外周血是比较可行的。     

HBL-3 Cell Line,Derived from Precursor B-cell lymphoblastic Leukemia,Lacks Somatic Hypermutation of Immunoglobulin Heavy Chain Variable Region Genes

Somatic mutation (SM) analysis provides a useful tool for understanding the stages at which neoplastic differentiate from normal B-cells. B-cell precursor neoplasms are considered to be somatically premutational. However, the variable frequency of SM of the variable region (VH) genes has been described in cases of precursor B-cell acute lymphoblastic leukemia (PB-ALL). To better characterize PB-ALL based on the differentiation stage, we investigated the SM of the VH genes expressed by tumor cells of the surface immunoglobulin (sIg)^– HBL-3 cell line derived from childhood PB-ALL. In the HBL-3 cell line, the rearranged Ig heavy chain VH gene sequence showed no SM in the complementarity-determining regions of 1, 2, and 3, or in the framework regions of 1, 2, and 3 relative to the putative germline VH gene sequences. In addition, the VH segment of HBL-3 cells showed no intraclonal sequence heterogeneity, indicating ongoing SM. Our data demonstrated that HBL-3 cells express unmutated and developmentally regulated rearrangement of VH genes at the stage of B-cell precursor cells. HBL-3 cells, which are derived only from the sIg^– PB-ALL, showed that SM cannot be recognized in VH genes of tumor cells before the expression of sIg.     

Detection and monitoring of clonality in peripheral blood and bone marrow of patients with B-cell lymphoproliferative disorders

Bone marrow (BM) is accepted as the tissue of choice for the detection of monoclonal populations in leukemias and lymphomas; however, obtaining BM can be painful and traumatic for the patients. Although it is possible to detect clonality in peripheral blood (PB) samples, there are no reports comparing the results observed from BM with those from PB. Lymphoblastic leukemias and lymphomas are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementarity Determining Regions (CDR). Of these, CDR3 is unique for each lymphocyte and therefore it can be used as a tumour-specific marker in these malignant disorders. Among the 104 patients from whom we obtained pre-treatment paired samples of PB and BM, 94 (90.4%) showed concordant results. Similarly, at the end of treatment, 40 of 44 patients (90.9%) showed this concordance. During treatment only 24 patients were monitored and monoclones disappeared in 12 patients; in the other half, they persisted either partial or totally. We demonstrate that the detection and monitoring of monoclonal populations in the PB, in comparison with BM, was achieved with a statistical sensitivity of 90% and specificity of 92%.     

Detection of minimal residual disease in B Chronic Lymphocytic Leukemia (CLL)

In the absence of specific chromosomal translocations the best method for detecting minimal residual disease (MRD) in B cell malignancies is based on the uniqueness of immunoglobulin (Ig) genes rearrangement. We here report a very sensitive method for assessing MRD in complete hematological remission (CHR) chronic lymphocytic leukemia (CLL) patients as defined by the international workshop on CLL (IWCLL). Patients: Twelve CLL patients in CHR and complete phenotypic remission (CPR) were included in the study. Eight of them received Fludarabine (FDR), one was treated by Chop regimen, and the remaining 3 were rescued by polychemotherapy followed by autologous bone marrow transplantation (ABMT). Methods: DNA extracted from peripheral blood lymphocytes (PBL) of each patient was amplified with VH family specific and framework 3 primers in 5 and a consensus JH primer in 3, before treatment and sequentially after the CPR completion. When no clonal rearrangement could be detected by this assay, the CDR3 sequence specific probe of the clone was used as the 3 primer, associated to the VH family specific primer in 5. PCR products were analyzed by classical procedures in agarose and/or acrylamide gels. Results: Mixtures of leukemic cells and normal PBL showed detection of a single leukemic cell among more than 10⁵ normal cells. Four out of the 12 patients achieved molecular remission (MR) when employing CDR3 amplifification. All 3 autografted patients were in MR, whereas only one out of the 9 patients treated by chemotherapy alone achieved MR. When using a clone specific probe, a clonal signal was observed in all cases but one (ABMT). Results presented here confirm that MR may be achieved in a few cases of B-CLL. Further studies are needed to determine the exact relationship between MRD and clinical outcome.This work was supported by a grant from Schering S.A.     

Chronic myeloid leukaemia lymphoid blast crisis. Relevance of molecular analysis at the bcr and immunoglobulin heavy chain gene level in monitoring response to therapy and residual disease

Southern blot analyses were performed in sequential DNA samples from 4 patients with Ph' + chronic myeloid leukaemia (CML) who underwent lymphoid or mixed blast crisis (BC). Genomic rearrangements at the breakpoint cluster region (bcr) and immunoglobulin heavy chain (IgH) gene level provided, in these cases, a sensitive and specific evaluation of response to therapy both in terms of blasts and Ph' + cell suppression. Recurrent BC was molecularly characterized in the 4 patients, showing each time identical individual specific DNA rearrangement patterns. Residual blasts were detected in 2 cases during intervening chronic phases by IgH rearrangements. Such findings highlight the specificity of these molecular markers, clearly indicating the failure of ablative therapy in eradicating the neoplastic clone. Finally, molecular and phenotypic identity in individual recurrent BC also suggested, in our cases, a lack of clonal evolution during disease progression.