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。     

Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype

Anaplastic large cell lymphoma (ALCL) is a distinct entity of non-Hodgkin lymphoma, characterized by a proliferation of pleomorphic large lymphoid cells that express CD30. Recent studies have found that a subset of ALCL aberrantly expresses a chimeric anaplastic lymphoma kinase (ALK) protein as a result of t(2;5)(p23;q35) or variant translocations. ALK-positive ALCLs feature good prognosis, but some of them lead to poor outcomes. Since CD56 is expressed in some ALCLs, its clinical significance was examined in a series of T/null cell type ALCLs. Of 143 patients, 83 (58%) showed ALK-positive staining, and of 140 patients, 25 (18%) expressed CD56. The ALK-positive subgroup was characterized by a younger age of onset (P <.0001), lower serum lactate dehydrogenase level (P =.01), better performance status (P =.03), less frequent extranodal involvement (P =.01), lower international prognostic index (IPI) categories (P =.002), and superior survival (P =.0009) in comparison with the ALK-negative group, suggesting that ALK is a specific marker defining a distinct subtype. CD56(+) cases showed a significantly poor prognosis overall (P =.002) as well as in both ALK-positive and ALK-negative subgroups (P =.02 and P =.04, respectively). Multivariate analysis confirmed that CD56 is independent of other prognostic factors, including IPI. Although CD56(+) cases showed a higher incidence of bone involvement, no other differences in clinicopathologic parameters were found between the CD56(+) and CD56(-) groups. These findings suggest that CD56 is not a marker to identify a distinct subtype of ALCL, but a strong clinical prognostic factor. Effective therapeutic approaches should be explored for high-risk ALCL patients, who can be identified by means of a prognostic model, including CD56.     

Comparison of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients

Purpose

The detection of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients is an independent prognostic factor. In recent years, the focus of research was on finding methods for the detection of circulating tumor cells (CTC) in peripheral blood (PB). In this study, we investigated the presence of DTC-BM and CTC by simultaneous examinations in 202 patients at different stages of the disease.

Methods

Immunocytochemical examination of DTC-BM (10–20 ml of BM) with the anti-cytokeratin (CK) antibody A45B/B3 followed a standardized protocol. Analysis of PB (7.5 ml) for the presence of CTC was performed with the CellSearch Analyzer system (Veridex, Raritan, NJ, USA).

Results

Overall, DTC-BM were detected in 57/202 (28.2 %, 1–>1,000 DTC) and CTC in 41/202 (20.3 %, 1–411 CTC) patients. Congruence of findings was 71.3 % (144/202, p = .002). In 147 pts with primary diagnosis, congruence of results was 69.4 % (102/147). There was no significant correlation between DTC or CTC and the established pathological factors. After a median follow-up time of 34 months (0–82), presence of CTC was borderline significant for tumor-associated death (p = .060). For 41 patients at recurrence-free follow-up, congruence of results was 75.6 % (31/41, p = .018). In this group, there was a patient with both the highest DTC (>1,000) and CTC (411) count, and she presented with distant metastases 3 months later and had died 5 months after that. Of 14 patients with metastatic disease, 9 showed both DTC and CTC (overall congruence 78.6 %, p = .176).

Conclusion

There was significant congruence between DTC and CTC, which even increased in patients at follow-up and in those with metastases. Repeated CTC examinations could be a valuable tool for monitoring patients or the effectiveness of therapies.     

CMV monitoring after peripheral blood stem cell and bone marrow transplantation by pp65 antigen and quantitative PCR.

We prospectively monitored 74 consecutive allogeneic and 50 autologous patients after bone marrow/stem cell transplantation from May 1999 to October 2000 at our institution with quantitative CMV PCR and pp65 antigen assay once weekly from conditioning therapy to days 120 and 80 after transplantation, respectively. Written informed consent was obtained from every patient. CMV prophylaxis consisted of acyclovir during transplant. Additionally all patients received only platelet products from CMV-negative donors. In the case of CMV infection preemptive therapy with gancyclovir was applied. In the case of CMV disease high-dose immunoglobulin was given as well. In the allogeneic setting 16 out of 74 (22%) patients developed a positive PCR. Seven episodes of a positive pp65 antigen assay occurred in six allograft recipients. In the autologous setting no positive assay was found during the whole observation period. Additionally, in 6/16 patients a lymphoproliferative assay was performed during CMV infection. Two patients showed a positive (15 and 5.4) and four a negative (2,1.6,1,1.8) stimulation index.     

直肠癌患者外周血循环肿瘤细胞围手术期变化及临床意义

［摘要］　目的　探讨直肠癌患者围手术期外周血循环肿瘤细胞（CTCs）的变化情况及其对患者预后的影响。方法　选择2015-09～2016-12广西壮族自治区人民医院胃肠外科收治的直肠癌患者30例,采用随机数字表法将其分为腹腔镜手术组和开腹手术组,每组15例。采用CanpatrolTM 2代技术对患者手术前后的外周血CTCs进行分型检测。术后随访22～33个月,分析两组患者手术前后的CTCs水平变化,术前各分型CTCs与疾病特征的关联性以及手术前后CTCs对患者预后的影响。结果　腹腔镜手术组术后总CTCs水平较术前显著降低（P<0.05）。腹腔镜手术组术前混合型CTCs水平显著高于开腹手术组（P<0.05）,但术后两组差异无统计学意义（P>0.05）。两组手术前后间质型CTCs和上皮型CTCs水平比较差异均无统计学意义（P>0.05）,且同组手术前后比较差异亦无统计学意义（P>0.05）。间质型阴性组肿瘤直径≤3 cm的人数比例显著高于间质型阳性组（P<0.05）。术前总CTCs、混合型CTCs、上皮型CTCs情况与患者年龄、性别、T分期、N分期及肿瘤直径的关联性不显著（P>0.05）。3例术后CTCs阴性者在随访过程中均未发生死亡;27例术后CTCs 阳性者在随访过程中出现死亡4例,术后CTCs阴性者的生存情况显著优于阳性者（log-rank检验： χ²=6.553,P=0.031）。2例术前CTCs阴性者在随访过程中均未死亡,28例术前CTCs阳性者在随访过程中出现死亡4例,术前CTCs阴性者的生存情况显著优于阳性者（log-rank检验： χ²=5.245,P=0.027）。结论　外周血CTCs可用于直肠癌患者预后评价,指导临床治疗。     

Monitoring gastric lymphoma in peripheral blood by quantitative IgH allele-specific oligonucleotide real-time PCR and API2-MALT1 PCR

Gastric extranodal marginal zone lymphoma (EMZL) often shows prolonged localised disease, but the present study demonstrated the presence of tumour cells in peripheral blood (PB) of low stage patients. We studied the presence of tumour cells in PB in gastric lymphoma patients harbouring or lacking t(11;18)(q21;q21), by real-time immunoglobulin (Ig)H allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) and API2-MALT1 PCR. Tumour cells were exclusively detected in PB of t(11;18)(q21;q21)+-EMZL patients. The presence of tumour cells in PB and gastric biopsy follow-up samples showed a good correlation in these patients, suggesting clinical relevance for monitoring of tumour cells in PB of gastric t(11;18)(q21;q21)+-EMZL patients.     

The significance of host haemopoietic cells detected by cytogenetic analysis of bone marrow from recipients of bone marrow transplants

Bone marrow from 39 patients who received a bone marrow transplant (BMT) from a matched donor of different sex were studied by chromosome analysis for evidence of mixed haemopoietic chimaerism (MC). Recipient metaphases were detected in the bone marrow of 10 patients after BMT. Patients in whom MC was detected within 6 weeks of BMT did not all have a poor outcome. Two of seven are disease-free survivors at greater than 470 and greater than 632 d. All three patients in whom MC was diagnosed more than 6 weeks after BMT subsequently relapsed. Four factors appear to be important in determining the probability of relapse when MC is detected in a patient after BMT: the timing of detection of residual recipient cells; the proportion of these cells in the bone marrow; persistence of these cells in increasing proportions; and the karyotype of the recipient metaphases detected. Cytogenetic assessment may provide the earliest indication of relapse in these patients. In addition, this study provides further evidence that cyclophosphamide and total body irradiation, as used in these patients, may be inadequate conditioning therapy for BMT.     

Anaplastic lymphoma kinase activity is essential for the proliferation and survival of anaplastic large-cell lymphoma cells

The roles of aberrant expression of constitutively active ALK chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) have been well defined; nevertheless, the notion that ALK is a molecular target for the therapeutic modulation of ALK+ ALCL has not been validated thus far. Select fused pyrrolocarbazole (FP)-derived small molecules with ALK inhibitory activity were used as pharmacologic tools to evaluate whether functional ALK is essential for the proliferation and survival of ALK+ ALCL cells in culture. These compounds inhibited interleukin 3 (IL-3)-independent proliferation of BaF3/NPM-ALK cells in an ALK inhibition-dependent manner and significantly blocked colony formation in agar of mouse embryonic fibroblast (MEF) cells harboring NPM-ALK. Inhibition of NPM-ALK phosphorylation in the ALK+ ALCL-derived cell lines resulted in significant inhibition of cell proliferation and induction of apoptotic-cell death, while having marginal effects on the proliferation and survival of K562, an ALK- leukemia cell line. ALK inhibition resulted in cell-cycle G1 arrest and inactivation of ERK1/2, STAT3, and AKT signaling pathways. Potent and selective ALK inhibitors may have therapeutic application for ALK+ ALCL and possibly other solid and hematologic tumors in which ALK activation is implicated in their pathogenesis.     

Flow cytometric detection of circulating tumour cells in nucleophosmin/anaplastic lymphoma kinase-positive anaplastic large cell lymphoma: comparison with quantitative polymerase chain reaction

Quantification of occult circulating tumour cells in blood or bone marrow (BM) enables the identification of patients with a high risk for relapse in nucleophosmin/anaplastic lymphoma kinase (NPM-ALK)-positive anaplastic large cell lymphoma (ALCL). We have developed a flow cytometric (FCM) assay to quantify the rare ALK- and CD30-positive ALCL cells. When ALCL cells were admixed with normal peripheral blood or BM, ALK- and CD30-positive cells could be detected above background level at an added concentration of 10(-5) for all three cell lines tested. Sensitivity and costs of the assay were compared with quantitative real-time polymerase chain reaction (PCR) for NPM-ALK. The results of the FCM assay and quantitative PCR for NPM-ALK correlated. The sensitivity of the PCR exceeded that of the FCM by at least one log. Quantitative PCR was more time-consuming and expensive than FCM. Both methods were compared on BM or blood samples from 11 ALCL patients. FCM using antibodies against ALK and CD30 can sensitively and specifically detect the circulating ALCL cells in BM or blood. This method needs to be tested in a larger cohort of patients to determine whether it has sufficient sensitivity to be used as a substitute for quantitative PCR.     

Novel PCR primers to diagnose visceral Leishmaniasis using peripheral blood,spleen or bone marrow aspirates

Objective: To establish a suitable method of diagnosis of visceral Leishmania sis(VL) using peripheral blood, spleen or bone marrow aspirates. Methods: Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients(n=26). A new PCR primer pair(MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. Results: The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. Conclusions: The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.     

A quantitative study of peripheral blood stem cell contamination in diffuse large-cell non-Hodgkin's lymphoma: one-half of patients significantly mobilize malignant cells

Autologous transplantation using peripheral blood stem cells (PBSCs) collected after chemotherapy, followed by growth factor administration (ASCT), is increasingly used in the treatment of non-Hodgkin's lymphoma (NHL). However, quantitative data regarding contaminating malignant cells in the harvests are still scarce. We prospectively investigated 37 diffuse large-cell lymphomas (DLCLs) in complete remission (CR) that were treated according to multicentric protocols at our centre. DNA was extracted from the diagnostic lymph node. The complementarity-determining region (CDR) III was sequenced and a patient-specific oligomer synthesized. Contamination was evaluated semiquantitatively by polymerase chain reaction (PCR) and was confirmed by a limiting dilution analysis. PBSCs collected at regeneration after administration of granulocyte colony-stimulating factor (G-CSF), steady-state bone marrow (BM) and peripheral blood samples at CR were compared. DNA was available in 37 patients, from which 22 rearrangements could be sequenced. Patients (n = 15) who had both the required follow-up samples and a suitable clonal marker were investigated. In two cases, the patient-specific PCR assay set up at diagnosis later gave false-negative results in samples in which clonal DNA was still detectable by other sets of primers. PBSC contamination was highly variable: 7 out of 15 patients showed a PBSC/BM ratio of NHL cells greater than 1 log, whereas 8 out of 15 patients showed no difference and could vary from one apheresis to another. Eight ASCTs were performed, five of which used highly contaminated PBSCs: four patients relapsed early, three with disseminated lymphoma. Thus, 50% of DLCLs in CR seem to mobilize significantly malignant cells at regeneration under G-CSF. Considering the higher numbers of cells reinfused, this translates into a much higher number of lymphoma cells reinfused when compared with autologous bone marrow transplantation (ABMT). However, their clonogenic potential remains unknown and, despite concerning observations in certain cases, it is still unclear whether this has an impact upon the outcome of ASCT.     

Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is an aggressive lymphoma that is frequently associated with the t(2;5)(p23;q35), resulting in expression of a fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which can be detected by either monoclonal or polyclonal antibodies to the ALK protein. The clinical features of adults with ALCL are incompletely described, and the prognostic factors that are useful for predicting survival remain unclear. This report describes the clinical and laboratory findings in 70 adults with systemic ALCL who were treated with curative intent. We attempted to identify the clinical and pathological factors of prognostic importance, including the International Prognostic Index (IPI), immunophenotype, and expression of the ALK protein. The median age of the patients was 49 years (range, 15 to 75). There were 26 women and 44 men with a median follow-up of 50 months for living patients. Advanced stage was present in 56% and B symptoms were noted in 70% of the patients. Immunostains showed that 46% of the cases had a T-cell phenotype, 36% a null phenotype, and 18% a B-cell phenotype. The expression of ALK protein was found in 51% of the cases. The IPI factors were evenly distributed between the ALK+ and ALK- groups, except that the ALK+ patients were younger (median age, 30 v 61 years; P <.002). The ALK+ cohort included cases with null (44%), T-cell (42%), and B-cell (14%) phenotypes. All 10 cases with cytogenetic or molecular evidence of a t(2;5) were ALK+. The 5-year overall survival (OS) of the entire cohort was 65%. The 5-year OS of the ALK+ and ALK- cases was 79% and 46%, respectively (P <.0003). Analysis of only the T-cell/null cases (n = 57) showed a 5-year OS of 93% for the ALK+ cases and only 37% for the ALK- cases (P <.00001). Univariate analysis of the clinical features showed that age 相似文献