Removal of tumour cells from bone marrow: an evaluation of the available techniques

Autologous bone marrow transplantation has offered a way of increasing the dose of drugs and radiotherapy which can be used to treat patients with malignant disease in an attempt to eradicate tumour. Bone marrow is taken prior to treatment and returned to the patient to 'rescue' haemopoietic function after ablative therapy is completed. Bone marrow removed for autograft may be contaminated with tumour cells at the time of harvest, and it is undesirable to return these to patients even though there are little data available concerning the number of tumour cells necessary to reseed various malignancies. This review considers the various methods available for removing tumour cells from bone marrow destined for autologous transplantation, and evaluates their advantages and disadvantages.     

An effective,direct immunomagnetic procedure for purging acute lymphoblastic leukemia cells from human bone marrow

The AB4 monoclonal antibody, which recognizes an HLA-DR epitope, was found to bind to a high percentage of malignant blast cells in samples obtained from 27 patients with ALL. These included 11 of 11 cases with c-ALL, 3 of 7 with pre-pre-B, and 8 of 9 cases with pre-B ALL. AB4 was used together with anti CD10 and anti CD19 antibodies and super-paramagnetic particles for developing a direct immunomagnetic procedure for purging human bone marrow of leukemic cells. In model experiments with KM3 cells admixed to mononuclear bone marrow cells, the individual antibodies each removed 2.8–3.1 logs and 3.6–4.1 logs of tumor cells with one and two purging cycles, respectively. In comparison, the efficacy of a mixture of the three antibodies was 4.4 logs with one treatment cycle, and > 5 logs with repeated treatments. Whereas the use of a commercially available anti-HLA-DR antibody resulted in a 90% reduction in the survival of CFU-GMs and normal blast colonies, AB4 had only a moderate effect on the progenitor cells (46% and 30% reduction). In conjunction with autologous transplantation, bone marrow from a patient was purged with the antibody mixture and 50% of the CFU-GMs and 47% of the CD34⁺ cells remained after treatment. The patient showed a normal engraftment, reaching a level of 0.5 × 1071 neutrophils by day 20 and 20 × 107 9/1 platelets by day 30. It is concluded that the antibody cocktail may safely and effectively be used for rapid autograft purging in patients with c-ALL, and also in phenotypically selected cases with other subtypes of ALL. This work was supported by the Norwegian Cancer Society     

Monitoring the remission induction therapy of acute nonlymphoblastic leukemia by bone marrow cell culture criteria

In 31 cases of acute nonlymphoblastic leukemia, bone marrow cells were serially cultured in semi-solid agar during the remission induction therapy. A normal in vitro cell growth pattern returned in 15 out of 22 patients up to 77 days before a complete remission was established by clinical and hematological criteria. In 6 cases the return of normal colonies coincided with clinical and hematological evidence of a complete remission. Nine patients failed to attain a remission and died from complications of bone marrow aplasia. Only one had a normal number of colonies and a normal cluster/colony ratio in cultures prepared 11 days after the completion of the first course of chemotherapy. At this time, his platelet count increased to normal level, possibly indicating a developing remission. Bone marrow cell culture criteria are useful in monitoring the remission induction therapy in patients with acute nonlymphoblastic leukemia. An early return of normal in vitro cell growth pattern suggests an approaching remission, which may be achieved several weeks later.     

Studies of myelopoiesis in vitro on blood and bone marrow cells of patients with acute leukemia in long-term remission

In vitro myelopoiesis in a group of ten patients with acute leukemia (6 AML, 1 APML, 3 ALL) in long-term remission has been studied. The patients remained in first stable remission for at least 4 years and maintenance therapy had been completed in all patients except one. In the patients, colony formation of bone marrow cells after 7 days of incubation was significantly increased compared to a representative control group. The proliferation of microclusters after 3 incubation days was also markedly enhanced, and accelerated proliferation of all aggregates (microclusters, macroclusters and colonies) could be demonstrated in culture with the patients' bone marrow cells. The use of autologous feeder layers and autologous serum showed no inhibitory effect on colony formation. The proportion of eosinophil colonies formed with the patients' bone marrow cells was in the normal range. In contrast to the high proliferation capacity of bone marrow precursor cells the CFU-c number of peripheral blood was significantly decreased in the patients' group. No significant correlation between CFU-c number of bone marrow and blood cells could be found. The colony stimulating activity of the patients' peripheral mononuclear cells was normal compared to healthy controls. We conclude from this study that even in long-term remission of acute leukemia certain in vitro abnormalities exist in myelopoietic proliferation and regulation.     

The incidence of recurrence of leukemia in donor cells after allogeneic bone marrow transplantation

A total of 243 patients with acute leukemia received a marrow graft from a donor of the opposite sex. Seventy-five patients subsequently relapsed, and specimens of the recurrent leukemia cells were available for cytogenetic analysis in 54. Three relapses were in donor-type cells. Four patients whose relapses were in host-type cells showed a cytogenic abnormality that differed from the original leukemic clone. We conclude that a ‘new’ leukemia in donor cells, and possibly in host cells, constitutes a small but significant fraction of recurrent leukemia after marrow grafting.     

Relative and absolute deficiency of bone marrow endogenous colony stimulating activity in acute myeloid leukemia: Relation to response to chemotherapy

The purpose of this study was to determine possible mechanisms for the recently observed association between insensitivity of acute myeloid leukemia (AML) clonogenic cells to colony stimulating activity (CSA) and poor response to induction chemotherapy. The bone marrow endogenous CSA was determined using semi-solid agar cultures by measuring the response of the AML patients' own clonogenic cells to endogenous CSA. The results show that whereas 31% ($\text{5}\text{16}$) of patients at presentation have deficient bone marrow endogenous CSA production over 50% ($\text{11}\text{21}$) have relative deficiency of endogenous CSA, due to insensitivity of the patients' clonogenic cells to CSA.Although there is an association between relative deficiency of endogenous CSA and a poor response to therapy the relationship is not close enough to explain the previously observed highly significant correlation between insensitivity to CSA and poor response to therapy. The results suggest that two independent mechanisms are operative in the association between the CSA-insensitive phenotype and poor response to therapy, one via the tendency to relative endogenous CSA deficiency in the CSA-insensitive group and another via some additional feature of these poor response AML phenotypes which is independent of the presence or absence of endogenous CSA deficiency.     

细胞芯片法在急性白血病骨髓样本免疫分型中的应用

目的:应用急性白血病免疫分型诊断的细胞芯片对急性白血病患者的骨髓样本进行免疫分型,从而快速诊断白血病的类型.方法:将细胞悬液滴于细胞芯片,那些有相应CD抗原的细胞只能与相应的单克隆抗体的点结合,进行Wright's染色,并对65例临床白血病骨髓样本进行分型并验证结果.结果:本实验所得的免疫分型结果与流式细胞仪的白血病免疫分型结果一致.结论:本实验使用的细胞芯片可作为临床白血病免疫学分型的诊断依据.     

Cryopreserved autologous bone marrow infusion following high dose chemotherapy in patients with acute myeloblastic leukemia in first relapse

Thirteen patients with AML in first relapse were treated with high dose combination chemotherapy followed by cryopreserved autologous bone marrow transplantation (ABMT). The first four patients received the COATA-Roma regimen, consisting of CTX, VCR, CA, 6-TG and ADM; nine additional patients received the BAVC regimen consisting of BCNU, AMSA, VP-16 and CA. A median of 1.6 X 10(8) fractionated nucleated bone marrow cells/kg body weight were reinfused. The median of GM-CFU-C recovered was 4.7 X 10(4)/kg. Out of 13 patients, 10 (76.9%) achieved CR, 3 had profound aplasia and died from hemorrhagic or infectious complications. Of the 10 patients who achieved CR, 1 died after 1 week from heart failure, 5 relapsed respectively 17, 20, 21, 21, 42, weeks after ABMT, 4 are still in CR after 2+, 14+, 17+, and 120+, weeks. Of the 9 patients treated with BAVC regimen, 8(88.8%) achieved CR. Four patients relapsed after a median of 19.7 weeks and 4 are still in complete remission. Of interest is the fact that the second complete remission of one patient is longer than the first one, despite the fact that marrow was not purified by any in vitro treatment. In conclusion we can say that BAVC regimen is highly effective in obtaining second complete remission in patients with AML and prolonged disease free survival can be achieved at least in a small number of cases.     

Proliferation of normal and leukemic Tdt+ bone marrow cells in man

Proliferative characteristics of lymphoid cells with detectable amounts of nuclear terminal deoxynucleotidyl transferase (TdT) in normal or regenerating non-leukemic bone marrow of children were assessed by sequential immunological and cytokinetic studies on single cells and compared to those of TdT⁺ cells in the bone marrow of children with non-T, non-B acute lymphoid leukemia (ALL) at diagnosis or in remission. The median labelling index (LI) of non-leukemic TdT⁺ cells was 21.3% (range 13.2 to 29.7%). The LI of non-leukemic TdT⁺Ia⁺ cells (range 18.2 to 32.6%) was always higher than that of non-leukemic TdT⁺Ia⁺ cells (range 0 to 5.3%). Leukemic TdT⁺ bone marrow cells of children with previously untreated non-T, non-B ALL had a LI significantly (p < 0.001) lower (median 6.1%; range 1.4 to 19.7%) than the LI of non-leukemic TdT⁺ cells. In patients with non-T, non-B ALL in remission neither the percentage of TdT⁺ cells nor the LI of TdT⁺ cells appeared to be useful for detecting early relapse.     

CFU-C abnormalities and cytochemical defects in bone marrow of adult patients during remission of acute leukemia

Twenty-three adult patients in complete remission (CR) from acute leukemia were investigated for the steady-state bone marrow (BM) CFU-C concentration and the cytochemical findings of neutrophils and monocytes. There was considerable variation in CFU-C concentrations among patients. Patients with abnormality high or low values tended to relapse earlier. Repeated assays revealed constant CFU-C values in individual long-term cases of remission (4 cases). One case of M4 (myelomonocytic) and the case of M5b (monocytic) leukemia revealed a complete lack of nonspecific esterase activity in CR monocytes as well as initial leukemic promonocytes; they also showed abnormally high or low concentrations of CFU-C and relapsed early. This finding suggests that hematopoiesis in CR bone marrow occurs from abnormal stem cells common to initial, acute leukemic clones in such cases.     

Ex vivo elimination of lymphoblastic leukemia cells from human marrow by mafosfamid

Studies were performed to evaluate the anti-tumor activity of mafosfamid, a new synthetic derivative of cyclophosphamide. We tested its ability to eliminate lymphoblastic leukemia cells from autologous bone marrow grafts following a 30 min preincubation in a highly sensitive clonogenic assay. Treatment with 50-100 micrograms mafosfamid/ml eliminated more than 4 logs of contaminating clonogenic tumor cells from a 200-fold excess of normal bone marrow. Flow cytometric studies showed differences in cell cycle kinetics between mafosfamid-resistant and mafosfamid-susceptible tumor cell clones. Compared to drug susceptible clonogenic tumor cells, clones that resisted treatment with 100 micrograms mafosfamid/ml exhibited a smaller percentage of cells in S-phase, indicating that mafosfamid is mostly cytotoxic to rapidly cycling tumor cells. The combination of mafosfamid and a target cell selective immunotoxin containing pokeweed anti-viral protein was superior to mafosfamid alone or immunotoxin alone for purging mafosfamid-resistant leukemic cells from human marrow.     

Reinfusion of leukemic cells with the autologous marrow graft: preclinical studies on lodging and regrowth of leukemia

To evaluate in quantitative terms the contribution of leukemic cells present in the autologous marrow graft to the occurrence of leukemia relapse after autologous bone marrow transplantation, preclinical studies were performed in a rat model for human acute myelocytic leukemia (BNML). Firstly, the number of leukemic cells which--after intravenous transfer--cause death from leukemia in 50% of the recipient rats proved to be 24.7 cells. Secondly, it appeared that the regrowth of leukemic cells in rats heavily pretreated with high-dose cyclophosphamide and total body irradiation was significantly hampered as compared with non-pretreated controls as judged by survival times (37 and 31 days, respectively after 10(3) BNML cells i.v.). The most likely explanation is treatment-induced damage to the microenvironment. Differences in patterns of lodging of infused leukemic cells were ruled out by comparing the uptake of 51Cr-labeled BNML cells in various organs. Finally, extrapolated from the available rat data on log leukemic cell kill induced by high-dose chemoradiotherapy, an hypothesis is presented relating the total tumor load in man to the clinical outcome of autologous bone marrow transplantation. From this hypothesis it is derived that the minimal number of leukemic cells that causes leukemia upon intravenous transfer varies between 10(4) and 10(6).     

Increased in vitro radioresistance of bone marrow fibroblastic progenitors (CFU-F) from patients with acute non-lymphocytic leukemia

The proliferative potential following in vitro irradiation of bone marrow fibroblastic progenitors (CFU-F) derived from four patients with acute nonlymphocytic leukemia (ANLL) and seven nonleukemic subjects was compared. The CFU-F from the ANLL patients were significantly more radioresistant than the CFU-F from the nonleukemic subjects. The increased radioresistance in ANLL patients was evident in both the mean slope of the survival curve (control = ?0.385, ANLL = ?0.256) and in the D_o values (control = 2.68 Gy, ANLL = 4.61 Gy). Thus CFU-F derived from ANLL patients differ from those derived from nonleukemics in both radioresistance and in granulopoietic effects as suggested from previous studies.     

Characteristics of bone marrow and blood cells in human leukemia that produce leukemia inhibitory activity (LIA)

Cells in the bone marrow and blood of patients with acute and chronic myeloid and lymphoid leukemia which produce leukemia inhibitory activity (LIA) specific for normal granulocyte-macrophage progenitor cells (CFU-c) have been characterized as belonging to the third population of lymphoid-like cells which are neither T nor B but which have Fe receptors. The cells are non-adherent, non-phagocytic, of low density (<1.070 g/cm³), slowly sedimenting (2–6 mm/h) and present in the sheep red blood cell rosetting populations which are E^?, EAC^?, Ig^?, EA⁺, and are Ia^? as determined by a complement cytotoxicity test using rabbit anti-human Ia-like antibody. The LIA-producing cell has been further characterized by its response to certain agents with known immunoregulatory activity. Bacterial lipopolysaccharide, tuberculin purified protein derivative, Bacillus Calmette-Guérin, dextran sulphate and pokeweed mitogen suppressed the production of LIA. Total suppression of LIA production by LPS required the constant presence of this agent; but hydrocortisone and dexamethasone abrogated LPS suppression of LIA production. LIA could not be found in normal human adult bone marrow and blood cells or fetal bone marrow, spleen, or liver cells. Further evidence for the specificity of LIA action was obtained since LIA did not inhibit erythropoietin dependent human erythroid progenitor cell (BFU-e, CFU-e) proliferation.     

The separation of a mixture of bone marrow stem cells from tumor cells: An essential step for autologous bone marrow transplantation

KHT tumor cells were mixed with mouse bone marrow to simulate a sample of bone marrow containing metastatic tumor cells. This mixture was separated into a bone marrow fraction and a tumor cell fraction by centrifugal elutriation. Elutriation did not change the transplantability of the bone marrow stem cells as measured by a spleen colony assay and an in vitro erythroid burst forming unit assay. The tumorogenicity of the KHT cells was similarly unaffected by elutriation. The data showed that bone marrow cells could be purified to less than 1 tumor cell in more than 10⁶ bone marrow cells. Therefore, purification of bone marrow removed prior to lethal radiation-drug combined therapy for subsequent autologous transplantation appears to be feasible using modifications of this method if similar physical differences between human metastatic tumor cells and human bone marrow cells exist. This possibility is presently being explored.     

Risk of therapy-related myelodysplastic syndrome/acute leukemia following high-dose therapy and autologous bone marrow transplantation for non-Hodgkin's lymphoma.

BACKGROUND: Several recent reports have suggested that patients with non-Hodgkin's lymphomas (NHL) who undergo autologous stem cell transplantation (ASCT) are at increased risk of developing therapy-related myelodysplastic syndrome (tMDS) and acute myelogenous leukemia (tAML). PATIENTS AND METHODS: We analyzed 493 patients with NHL who underwent ASCT at The University of Texas M.D. Anderson Cancer Center between January 1990 and August 1999. RESULTS: With a median follow-up time of 21 months after HDT, 22 patients developed persistent cytopenia in at least one cell line with morphologic or cytogenetic evidence of tMDS or tAML. Univariate analysis identified prior fludarabine therapy, bone marrow involvement with lymphoma, and total body irradiation (TBI) as significant risk factors for the development of tMDS/tAML (P <0.05). Multiple logistic regression analysis showed that TBI was independently associated with an increased risk of developing tMDS/tAML (P <0.01). Further analysis of the patients who received TBI revealed that patients receiving TBI in combination with cyclophosphamide and etoposide were more likely to develop tMDS/tAML than those who received TBI with cyclophosphamide or thiotepa (P <0.01). The median survival of patients developing tMDS/tAML was 7.5 months (range 0-32 months). CONCLUSIONS: TBI, especially when used in combination with cyclophosphamide and etoposide as the pretransplant conditioning regimen, is a significant risk factor for the development of tMDS/tAML.     

自体骨髓移植联合MHC单倍体相合淋巴细胞治疗急性髓性白血病的临床疗效

目的:自体骨髓移植联合MHC单倍体相合淋巴细胞治疗急性髓性白血病的治疗效果和安全性。方法:以40例急性髓性白血病患者作为研究对象,随机分为两组各20例,研究组患者使用自体骨髓移植联合MHC单倍体相合淋巴细胞进行治疗,对照组单用自体骨髓移植进行治疗。结果:两组患者的造血系统都得到重建,重建时间以及并发症的发生均没有显著差异。而研究组患者复发率显著降低,且复发时间明显地长于对照组患者。研究组患者3年累积无病生存率明显地高于对照组患者,且差异具有统计学意义。结论:自体骨髓移植联合MHC单倍体相合淋巴细胞治疗能够有效控制急性髓性白血病病情,降低其复发,延长患者生存期。     

急性白血病骨髓间充质干细胞调控免疫的功能及机制

目的 探讨急性白血病患者骨髓间充质干细胞(MSC)的免疫原性及抑制异体T淋巴细胞增殖的功能和机制.方法 采用细胞贴壁法获取急性髓细胞白血病(AML)和急性淋巴细胞白血病(ALL)骨髓MSC,在低血清培养液中培养和扩增.采用流式细胞仪和免疫组化方法检测免疫表型,应用酶联免疫吸附实验检测MSC培养上清液中细胞因子的分泌水平,Transwell检测骨髓MSC抑制T淋巴细胞增殖的能力,混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力.结果 ALL骨髓MSC在倒置显微镜下为梭形,CD29、CD44和CD105的表达阳性率分别为98.81%、99.25%和90.52%,而CD31、CD34和CD45均为阴性.AML骨髓MSC表达CD29和CD44.ALL和AML骨髓MSC不表达人白细胞DR抗原(HLA-DR)和共刺激分子CD80、CD86和CD40.ALL骨髓MSC和AML骨髓MSC均具有分泌TGF-β1(567.58±52.64和357.15±33.52)、HGF(647.27±102.54和219.67±62.37)、IL-6(59.67±15.69和54.35±12.31)和IL-11(102.58±23.54和78.21±9.67)的功能,但是AML骨髓MSC的TGF-β1和HGF分泌水平明显低于ALL骨髓MSC(均P＜0.05).在MSC数量分别为0.5×104、1×104、2×104和5×104个时,加入AML骨髓MSC的CD3+T淋巴细胞3H-TdT掺入的CPM值分别为(3.58±0.54)×104、(2.87±0.33)×104、(1.78±0.51)×104和(1.15±0.15)×104,加入ALL骨髓MSC相应的CD3+T淋巴细胞3H-TdT掺入的CPM值分别为(1.96±0.31)×104、(1.57±0.28)×104、(0.91±0.41)×104和(0.22±0.11)×104,而未加入MSC的CD3+T淋巴细胞3H-TdT掺入的CPM值为(4.01±0.72)×104,AML和ALL骨髓MSC抑制T淋巴细胞的增殖存在明显差异.ALL骨髓MSC抑制T淋巴细胞增殖的能力可以被抗TGF-β1和HGF抗体逆转.结论 ALL骨髓MSC具有低免疫原性及体外调节免疫的功能,该免疫调节功能与其分泌细胞因子有关.AML骨髓MSC调控免疫的能力存在病理改变.

Abstract：

Objective To study the immunomodulatory effects and mechanisms of mesenchymal stem cells(MSC) derived from the bone marrow in acute leukemia patients in vitro. Methods Bone marrow mononuclear cells from acute myeloid leukemia(AML) and acute lymphoblastic leukemia(ALL)were obtained and cultured in low serum medium.The immunophenotypes were assessed by FACS and immunol histochemistry.The levels of cytokines were evaluated by enzyme linked immunosorbant assay (ELISA).T-cell suppression ability was evaluated by Transwell chamber assay.Moreover,the immunoregulatory ability of AML-and ALL-derived MSC was detected by mixed lymphocyte culture assay. Results ALL-derived MSC showed a typical fibroblast-like morphology.They were positive for CD29,CD44 and CD105,the positive rate were 98.81%,99.25% and 90.52%,respectively,while negative for CD31,CD45 and CD34.Moreover,ALL-and AML-derived MSC didn't express HLA-DR and costirnulatory molecules(CD40,CD80 and CD86ALL and AML derived MSC could secret several cytokines,such as TGF-β1(567.58 ±52.64 and 357.15 ±33.52),HGF(647.27 ± 102.54 and 219.67 ±62.37),IL-6(59.67 ± 15.69 and 54.35 ±12.31) and IL-11(102.58 ±23.54 and 78.21 ±9.67),the level of secretion of TGF-β1 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC.ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner,the counts per minute(CPM) were(3.58 ± 0.54) × 104,(2.87 ± 0.33) ×104,(1.78 ±0.51) × 104 and(1.15 ± 0.15) × 104 for AML derived MSC,and CPM were(1.96 ± 0.31)×104,(1.57 ±0.28) ×104,(0.91 ±0.41) ×104 and(0.22 ±0.11) ×104 for ALL derived MSC when MSCwere0.5×104,1×104,2×104 and5×104.In addition,the CPM was(4.01 ±0.72) ×104 in control group.The immunosuppressive ability was different between MSCs derived from AML and ALL.The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-βl and anti-HGF antibody.Conclusion ALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through cytokines.But MSCs derived from AML display abnormal changes in T-cell suppression ability.