Detection of hepatoma cells in peripheral blood of HCC patients by nested RT-PCR

AIM:To offer a more simple method with a high sensitivity and specificity for detection of hepatoma cells in peripheral blood of the patients with HCC.METHODS:Improved nested RT-PCR method was used to detect the expression of AFP mRNA in nuclear cells separated from peripheral venous blood.RESULTS:AFP mRNA contained in tenhepatoma cells was detected from 2mL peripheral blood.CONCLUSION:The improved nested RT-PCR assay for AFP mRNA expressed in cancer cells in peripheral blood might be a valuable method for clinical diagnosis of HCC.     

Release of peripheral blood stem cells following chemotherapy in childhood malignancies

Peripheral blood stem cells released following chemotherapy were examined in 21 children with malignancies in early remission. In order to obtain more than 1 x 10(5) CFU-GM per liter which is the minimal concentration to achieve autologous blood stem cell transplantation by cytapheresis, myelosuppressive chemotherapy which reduced leukocyte count below 1,000/microliters or neutrophil count below 200/microliters was necessary. Because the repetition of chemotherapy reduced the release of CFU-GM in peripheral blood, blood stem cells should be collected early after the beginning of chemotherapy. By long term culture method, peripheral blood stem cells seemed to contain pluripotent stem cells. Using our therapeutic protocol, more than 1 x 10(5) CFU-GM per liter could be obtained in malignant lymphoma and acute non-lymphoblastic leukemia, thus autologous blood stem cell transplantation seemed to be possible in these diseases.     

Detection of circulating gastric cancer cells in peripheral blood using real time quantitative RT-PCR

BACKGROUND/AIMS: Detection of occult cancer cells in peripheral blood or bone marrow has recently received a great deal of attention regarding the prediction of postoperative recurrence of the cancer, and for novel strategies of adjuvant therapy. This study addresses the detection of circulating tumor cells in peripheral blood in patients with gastric cancer using Quantitative RT-PCR. METHODOLOGY: Common mRNA targets for RT-PCR for detection of small numbers of cancer cells in gastric cancer are CK18, CK19, CK20, and CEA. Ten milliliter of peripheral venous blood was taken from 14 healthy Japanese volunteers and 101 patients with gastric cancer. Samples were analyzed using real-time TaqMan technology and a Model 7700-sequence system. The group of gastric cancer patients included 69 individuals with curative disease on preoperative diagnosis and 32 individuals with a non-curative operation or recurrence of the disease. RESULTS: The number of CK19 and CK20 mRNA copies was significantly increased in patients with a non-curative operation or recurrence of gastric cancer (CK19; p=0.0087, CK20; p=0.0022) compared with healthy volunteers. Cut-off levels of CK19 or CK20 copy numbers were determined by the maximum value of healthy volunteers. For CK19, there were 61 (88.4%) negative cases and 8 (11.6%) positive cases in 69 individuals with curative gastric cancer. There was a significant difference in tumor stages between CK19 positive and negative patients with curative disease on preoperative diagnosis. For CK20, there were 59 (85.5%) negative cases and 10 (15.5%) positive cases. There was no statistical difference between CK20 positive and negative cases for all clinicopathological factors. On postoperative day 14, there was a significant difference between positive and negative cases regarding tumor size, tumor stage, and lymph node metastasis for CK19, and tumor stage and lymph node metastasis for CK20. Five-year survival rates of patients with CK19 positive or negative cases were 50.0% or 79.0%, respectively (p=0.0347). While, for CK20, 5-year survival rates for positive cases was 51.9%, and for negative cases 78.9% (p=0.0490). CONCLUSIONS: Micrometastases of gastric cancer can be detected in circulating peripheral blood using quantitative real-time RT-PCR. CK19 is a better marker than CK18, CK20 and CEA, and could be clinically useful to estimate prognosis or to make a postoperative strategy of adjuvant treatment.     

Collection of allogeneic peripheral blood stem cells.

Circulating stem cells from healthy donors are increasingly used for allogeneic transplantation in patients with malignant lymphohaematopoietic disorders and solid tumours. Stem cells are collected by single or multiple aphereses following cytokine treatment. In the following, the effects of rhG-CSF on peripheral blood CD34+ cell and subset concentrations are described, as well as rhG-CSF related side-effects observed in normal donors. Safety considerations regarding cytokine treatment of normal donors are listed. The stem cell apheresis technology and cell yields are presented. Finally, novel aspects of stem cell mobilization, including the use of Flt3 ligand and anti-VLA4/VCAM-1 antibodies, are discussed.     

Detection of cancer stem cell like circulating tumor cells in peripheral blood of 45 patients with gastric cancer

<正>Objective To explore the expression and significance of CD44 gastric cancer stem cell biomarker positive circulating tumor cells(CTC)in peripheral blood of patients with gastric cancer.Methods From March 2010 to December 2012,45 patients with gastric cancer and 20healthy individuals were enrolled.Peripheral blood samples of all the subjects were collected.The expression of     

Allogeneic transplantation of peripheral blood stem cells.

Mobilized peripheral blood stem cells (PBSC) are increasingly being used instead of bone marrow for allogeneic transplantation. This chapter will briefly review important aspects of allogeneic PBSC transplantation including PBSC harvesting and donor safety, reconstitution of haematopoiesis and the immune system, graft-versus-host disease, graft-versus-leukaemia effects and graft engineering.     

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.     

Positive selection of autologous peripheral blood stem cells.

The development of monoclonal antibodies against differentiation antigens on human haematopoietic cells has led to a new concept in stem cell purification: the positive selection. In terms of autologous PBSC transplantation, the immature stem cells are identified by their expression of a specific antigen, the CD34. The CD34 antigen is expressed on early lymphohaematopoietic stem cells and progenitor cells, but not on mature blood cells or on tumour cells of several diseases. CD34+ cells are found in low numbers in bone marrow (<2%) and in even lower numbers in steady state blood (<0.01%) but may increase from 1 to 5% after mobilization using chemotherapy and/or growth factors. Several techniques have been set up to enrich PBSC grafts in CD34+ stem cells. The quality of each system is here analysed in terms of CD34 purity of the selected cell fraction, the CD34 cell recovery, the tumour cell depletion efficiency and the functional capacity ex vivo and in vivo of the selected cells. The final CD34+ cell purity of the selected fractions is correlated to the concentration of CD34+ cells before selection. The optimal recoveries and the highest purities were generally obtained when the initial CD34 content was roughly over 1%. Below this figure, the final purity seems to be less predictable. Besides the better tolerance resulting from the reduction in the number of autologous cells, and consequently the total volume of DMSO reinfused to the patient, the selective enrichment of the CD34 cell population offers a new approach to tumour purging. The procedure by itself results in elimination of about 99% in the total number of initial cells, thus allowing reduction of the overall tumour cell number in the final autograft. However, its major interest is that, in diseases where tumour cells do not express the CD34 antigen, it is theoretically able to completely eliminate the tumour contamination of the graft. Based on previous data showing that lymphoma, myeloma, neuroblastoma and breast cancer cells are not CD34+, pilot clinical trials for the separation and transplantation of CD34+ cells selected from PBSC of patients with these diseases have recently been conducted. The efficacy of CD34 selection in reducing the tumour load of the PBSC of patients with these diseases has been reported. However, the efficacy of purging may greatly differ between individual patients, and complete eradication of contaminating cells from PBSC grafts was not always reached. There is now evidence that purified CD34+ cells are capable of supporting haematopoietic reconstitution in autologous transplantation. However, until now no study has demonstrated clear evidence that the reduction of tumour cells from PBSC of patients by CD34+ cell selection resulted in a lower relapse rate post-transplant, as compared to unselected PBSC infusion.     

T-cell depletion of allogeneic peripheral blood stem cells.

The high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic peripheral blood stem cells (PBSC) to an elevated risk of acute and chronic graft-versus-host disease (GvHD). Thus, the use of an appropriate T-cell reduction or depletion technique might reduce this risk. The hazards of rejection and of a higher relapse rate should be avoided by maintaining a portion of the T-cells in the graft or by increasing the number of transplanted stem cells. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1,000-fold reduction of T-cells. Purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation. In transplantation from HLA-mismatched or three HLA-loci different family donors the amount of stem cells can be increased for reducing the incidence of rejection without increasing the T-cell number. In cases of poor marrow graft function a 'boost' with stem cells from the same family donor can be given. The risk of GvHD in transplantation from volunteer-matched unrelated donors might be reduced by T-cell depletion. If T-cells are used for enhancing the graft-versus-leukaemia effect, CD34+ enriched cells can be given for haematopoietic engraftment.     

小细胞肺癌血钙与外周循环肿瘤细胞的检测

目的研究不同分期小细胞肺癌患者血钙水平与外周血循环肿瘤细胞(CTC)的检测价值。方法选择我院84例小细胞肺癌患者为研究对象。根据国际肺癌的诊断标准对患者进行分期,局限期患者33例,广泛期患者51例。对所有患者血钙水平与外周血循环肿瘤细胞水平进行检测。结果患者的体内的血清钙以及游离钙在广泛期明显多于局限期,P0.05。患者的外周血循环肿瘤细胞≥1和≥5,广泛期明显多于局限期,P0.05。患者的血钙以及外周血循环肿瘤细胞水平会随病情发展而增加,P0.05各项指标和病情的发展程度呈现出正相关关系。结论综上所述,对小细胞肺癌患者体内血钙以及外周血循环肿瘤细胞水平的检测价值较高。对于患者的早期确诊,治疗以及病情的预后评价具有十分重要的意义。     

An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

Due to the lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories, an international multicenter trial was started with the participation of six laboratories (platforms: LightCycler LC, n=3; TaqMan TM, n=3). One hundred and eighty-six PB samples derived from healthy donors were spiked with serial dilutions (1:20 to 1:2x10(6)) of b2a2, b3a2 or e1a2 BCR-ABL positive white blood cells (WBC) from leukemic patients. After PAXgene stabilization, blinding, freezing and distribution, standardized RNA extraction, cDNA synthesis, PCR protocols and data evaluation were carried out. There was no significant difference in the results achieved using LC and TM technologies, but a considerable overall variation (CV=0.74 for ratios BCR-ABL/ABL). Up to a dilution of 1:1,000, 27/30 of the 2.5 mL samples tested positive. For higher dilutions, a PB volume of 5 or 10 ml was required to improve sensitivity. The study showed the feasibility of RQ-PCR standardization independent of the PCR machine used.     

Negative selection of autologous peripheral blood stem cells.

The use of chemotherapy and/or haematopoietic growth factor-mobilized peripheral blood stem cells (PBSC) has been shown to induce a more rapid haematopoietic recovery than the reinfusion of bone marrow (BM)-derived haematopoietic cells, thus reducing the morbidity and mortality of autologous stem cell transplantation (ASCT). PBSC collections were initially believed to have a lower incidence of tumour cells involvement than BM harvests. However, recent studies have shown that mobilized blood cell products of cancer patients eligible for autografting are frequently contaminated with tumour cells. Whereas positive selection of haematopoietic CD34+ stem cells has been largely used as a means of indirect purging of circulating CD34+ neoplastic cells, few groups have addressed the issue of tumour cell removal by direct targeting of cancer cells using physical or pharmacological strategies. In this chapter we review the available data concerning the contamination of tumour cells in PBSC collections from cancer patients, the functional and kinetic characteristics of primed CD34+ cells which may affect the haematopoietic toxicity of purging procedures developed to eliminate the minimal residual disease (MRD) from BM samples, and the preclinical and clinical results of the selective killing of residual tumour cells from leukaphereses. The limited amount of data published so far do not allow any firm conclusion on the clinical usefulness of purging protocols. Nonetheless, the successful extension of ex vivo purging to PBSC collections may improve the feasibility of randomized studies aimed at determining the importance of tumour-free autografts.     

Determination of peripheral blood stem cells by the Sysmex SE-9500.

The Sysmex SE-9500 automated haematology analyser provides an estimate of immature cells, referred to as 'haematopoietic progenitor cells' (HPC). The aim of this study was to evaluate the reliability and usefulness of the SE-9500 HPC parameter as compared with the CD34 + cell count and to determine whether the HPC count was of value in predicting the optimal harvesting time for peripheral blood stem cells (PBSC). Studies were performed on 112 samples from 21 patients with haematological malignancies and 13 healthy donors undergoing progenitor cell mobilisation. Coefficients of variation for the HPC count were 30%, 23.8%, 12.4% and 8.3% respectively for samples with low (4 x 106/l), medium (13 x 106/l), high (250 x 106/l) and very high (2413 x 106/l) counts. There was good linearity for HPC measurement in both peripheral blood (PB) and purified CD34 + cell suspensions (r > 0.995), and no detectable carryover was observed. There was an acceptable correlation between HPC and CD34 + cell counts for PB samples (r=0.669) and for CD34 + cell suspensions (r=0.859). Analysis of purified CD34 + cells using the SE-9500 HPC mode revealed that they appear both in the blast cell area and the immature granulocyte area of the analyser cell display. Quantitation of CD34 + cells and HPC during PBSC mobilisation showed good agreement between these parameters with regard to the optimal time for PBSC harvesting. These findings suggest that HPC counting with the Sysmex SE-9500 may be clinically useful for optimising the timing of PBSC collection.     

巢式RT-PCR技术检测肺癌患者外周血及淋巴结中微量转移癌细胞

目的　建立检测肺癌患者外周血及淋巴结中微转移癌细胞的敏感分子检测方法 ,并探讨其临床应用价值。方法　采用巢式反转录聚合酶链反应 (RT- PCR)技术特异引物扩增细胞角蛋白 19(CK19) m RNA,检测76例肺癌患者外周血及淋巴结中 CK19m RNA的表达情况。结果　 76例肺癌患者中 39例外周血 CK19m RNA阳性 ,阳性率为 5 1.3% (39/ 76 ) ,2 7例肺良性病变中仅 1例阳性 ,阳性率 3.7% ,15例健康对照者均为阴性。全部肺癌患者 15 7枚淋巴结中 CK19m RNA阳性率 38.9% (6 1/ 15 7) ,常规病理方法检测淋巴结阳性率 14 .6 % (2 3/15 7) ,两者相比 ,P<0 .0 5 ;HE染色阴性的 134枚淋巴结中 ,经巢式 RT- PCR技术检测证实存在癌转移的阳性率为 2 8.3% (38/ 134) ,两者相比 ,P<0 .0 5。结论　巢式 RT- PCR技术是一种特异性和敏感性均较高的微量癌细胞转移检测方法 ,可能有助于肺癌微转移的早期诊断