Development of a gene therapy based bone marrow purging system for leukemias

Although viable gene therapy based methods have been reported for the selective removal or purging of contaminating epithelial derived cancer cells from stem cell grafts, similar strategies for the purging of leukemia cells have been significantly less efficient. Hematopoietic cells are difficult targets for transduction with currently available vectors. Polylysine based molecular conjugate vectors (MCV) were previously found to effectively transduce both normal and malignant hematopoietic cells. A panel of human leukemia cell lines as well as CD34+ selected primary human hematopoietic progenitor cells (HPC) were tested for differential gene expression utilizing different promoters. Reporter gene expression under the control of RSV and SV40 promoters showed a 6-log fold increase in leukemia cells when compared to primary HPC. Using a polylysine based recombinant molecular conglomerate vector (recMCV) encoding the HSV-tk suicide gene under control of RSV, we demonstrated effective and specific cell killing in all leukemia cell lines as well as in primary human leukemia cells derived from chemotherapy refractory patients, while HPC survived under the same conditions at approximately 20% viability. These proof of principle experiments demonstrate that gene therapy technology could be utilized to successfully purge leukemia cells from HPC.     

Ex vivo differentiation therapy as a method of leukemic cell purging in murine bone marrow expansion cultures.

We investigated the use of differentiation therapy as a method of purging bone marrow (BM) of leukemic cells in ex vivo murine BM expansion cultures (delta-cultures). In clonal cultures and in suspension cultures a combination of the differentiation-inducing agents granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and all-trans-retinoic acid (ATRA) was found to be most effective in inducing the differentiation of the murine myelomonocytic leukemic cell line WEHI 3B D+ LacZ clone 2.8 (clone 2.8). Furthermore, we investigated the activity of a mutant form of IL-6, mutein, and found it to have a greater specific activity in cell proliferation assays and in a clone 2.8 differentiation assay than the native form of IL-6. Coculture of clone 2.8 and BM in IL-1 and kit-ligand-stimulated delta-cultures showed that the added stimuli, G-CSF, mutein, and ATRA, decreased the expansion of leukemic cells. Mice transplanted with G-CSF, mutein, and ATRA-purged BM had an increased survival time relative to nonpurged controls. The addition of G-CSF, mutein, and ATRA to delta-cultures did not result in any impairment of hematopoietic stem cells when measured 5 wk after transplantation.     

Immunological detection and definition of minimal residual neuroblastoma disease in bone marrow samples obtained during or after therapy

Immunological staining by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique has been used to recognize low levels of neuroblastoma cells in bone marrow mononuclear cells. Immunological phenotyping with 11 well characterized monoclonal antibodies was performed on 16 children with neuroblastoma and BM involvement during or after therapy. Neuroblasts were detected in 11 of 16 patients (0.1–5%), whereas BM biopsies on six of these patients were classified as normal. Aspirates, stained conventionally, were positive for pathological cells in three patients only.The comparison of the phenotype of the neuroblastoma cells at the time of diagnosis to the phenotype of the residual cells within one patient revealed differences. The phenotype of residual disease in different patients on the other hand showed a unique pattern. The above mentioned results lead to the conclusion that the immunological procedure is particularly suitable for the analysis of minimal residual neuroblastoma since the technique allows very minor cell populations to be identified in BM samples.     

Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma

The impact of bone marrow (BM) GD2-positive cells on survival has been evaluated in 145 Italian children with localised neuroblastoma (NB) evaluated at diagnosis by anti-GD2 immunocytochemistry. Nineteen of these (13.1%) were found to be BM GD2-positive, with the number of positive cells ranging between 1 and 155 out of 1 x 10(6) total cells analysed. Seven/19 (38.8%) GD2-positive vs 12/126 (9.5%) GD2-negative patients relapsed. The 5-year event-free survival (EFS) and overall survival of the GD2-positive patients was significantly worse than that of the GD2-negative ones (62.2 vs 89.9%, P<0.001; and 74.9 vs 95.9%, P=0.005, respectively). GD2 positivity was not associated to other known risk factors, and in particular to Myc-N amplification and 1p deletion. Among Myc-N-negative patients, the EFS of those negative for both GD2 and 1p deletion was significantly better than in children positive for either one of these two markers (EFS=96.9 vs 66.0%, P<0.001). In conclusion, GD2 positivity may represent a prognostic marker for patients with non-metastatic NB without Myc-N amplification, and its combination with genetic alterations might help identifying patients that require a more careful follow-up.     

Use of photosensitive, antibody directed liposomes to destroy target populations of cells in bone marrow: a potential purging method for autologous bone marrow transplantation.

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry.     

ECT骨显像和骨髓涂片在神经母细胞瘤骨转移中的临床应用

目的:探讨ECT骨显像和骨髓涂片在神经母细胞瘤骨转移的临床应用价值。方法:回顾性分析2011年10月至2018年10月我院收治的216例经病理组织、淋巴结或骨髓活检确诊为神经母细胞瘤患者的临床资料,收集同一时段内的ECT骨显像和骨髓涂片结果,分析两种方法在神经母细胞瘤骨转移的临床应用价值。结果:ECT骨显像阳性106例,ECT骨显像阴性110例,阳性率49.07%;骨髓涂片阳性84例,骨髓涂片阴性132例,阳性率38.89%。ECT骨显像阳性而骨髓涂片阴性组46例,占21.30%;ECT骨显像阴性而骨髓涂片阳性组24例,占11.11%;ECT骨显像及骨髓涂片均阳性组60例,ECT骨显像及骨髓涂片均阴性组86例,两种方法在诊断神经母细胞瘤患者有无骨转移的符合率为67.59%。在106例ECT骨显像阳性中骨髓涂片阳性有60例,骨髓转移阳性率为56.60%,说明ECT骨显像提示骨转移时已有56.60%患者发生骨髓转移。结论:ECT骨显像和骨髓涂片检查在神经母细胞瘤患者骨转移的临床分期、疗效和预后评估具有重要的临床意义,建议临床应同时行ECT骨显像和骨髓涂片检查,以提高神经母细胞瘤骨转移的检出率及准确率。     

Clinical potential of gene-directed enzyme prodrug therapy to improve radiation therapy in prostate cancer patients

Despite the advances in prostate cancer diagnosis and treatment, current therapies are not curative in a significant proportion of patients. Gene-directed enzyme prodrug therapy (GDEPT), when combined with radiation therapy, could improve the outcome of treatment for prostate cancer, the second leading cause of cancer death in the western world. GDEPT involves the introduction of a therapeutic transgene, which can be targeted to the tumour cells. A prodrug is administered systemically and is converted to its toxic form only in those cells containing the transgene, resulting in cell kill. This review will discuss the clinical trials which have investigated the potential of GDEPT at various stages of prostate cancer progression. The advantages of using GDEPT in combination with radiotherapy will be examined, as well as some of the recent advances which enhance the potential utility of GDEPT.     

Differential binding of soybean agglutinin to human neuroblastoma cell lines: potential application to autologous bone marrow transplantation

Normal human bone marrow cells were mixed with neuroblastoma cells from four different human cell lines, and the cell mixtures were separated by differential agglutination with soybean agglutinin (SBA). The unagglutinated cell fraction, previously shown to be highly enriched for the hematopoietic pluripotential stem cells and capable of reconstituting lethally irradiated adult patients with acute leukemia, was further fractionated by affinity chromatography on the lectin conjugated to Sepharose 6MB beads. Two independent assays, one using radiolabeling of the tumor cells and the other based on cloning of the neuroblastoma cells on agar, showed that the agglutination step alone removes 64-76% of the radiolabeled neuroblastoma cells and 85-98% of the clonogenic cells from the tumor/bone marrow cell mixture. Passage of the unagglutinated radiolabeled cells through SBA-Sepharose columns results in further purging of 28-53% of the neuroblastoma cells. Thus a combination of the two methods affords only one-log depletion for the neuroblastoma cells, compared to a three-log depletion achieved for a T-cell leukemia line CEM tested in parallel. It seems therefore that the agglutination technique, or the use of SBA-Sepharose columns, can be used only as a preliminary step for the purging of neuroblastoma cells from involved human bone marrow preparations. Staining with fluorescein isothiocyanate-conjugated SBA of nine different neuroblastoma cell lines, including the four tested in the fractionation studies, showed that more than 98% of the cells, of all the cell lines tested, specifically bind to the lectin, whereas no specific binding can be detected on the stem cell-enriched bone marrow cell fraction. However, the total number of receptors on the neuroblastoma cells is small compared to that of line CEM or normal granulocytes, which are strongly agglutinated by SBA. It seems therefore that the quantitative difference in the total number of SBA receptors is a crucial factor for purging by the agglutination technique or by affinity chromatography. Although these results show limitations to the use of both methods, this study establishes that all neuroblastoma cell lines tested express receptors for the lectin. Improved purging of neuroblastoma cells may possibly be achieved by targeting SBA-bound toxins or magnetic spheres to these receptors.     

In vitro purging of clonogenic leukemic cells from human bone marrow by heat: simulation experiments for autologous bone marrow transplantation.

In order to apply a simple purging method by heat to autologous bone marrow transplantation (ABMT), we have revaluated the ability to purge clonogenic leukemic cells from the simulated marrow mixture of normal marrow cells and leukemic cell lines (HL-60, Molt-3 and HEL) in vitro by heat, using two different clonogenic assays for normal granulocyte-macrophage progenitors (CFU-GM) and leukemic cell lines. It appeared that in vitro hyperthermia (42 degrees C for 120 min) is able to selectively remove clonogenic leukemic cells from simulated tumor cell-normal marrow mixtures even when leukemic cell concentrations are increased up to 3 x 10(6) cells/ml in vitro, and results in a 4-6 log destruction of clonogenic leukemic cells/ml according to a limiting dilution assay, while leaving half of normal CFU-GM surviving. The hyperthermic purging of clonogenic leukemic cells was not affected in the presence of normal marrow cells in vitro. This high level of clonogenic leukemic cell depletion by heat correlated with that of immunologic and pharmacologic studies. These results suggest that in vitro hyperthermia could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous marrow grafts before ABMT.     

Antibody-directed enzyme prodrug therapy: pharmacokinetics and plasma levels of prodrug and drug in a phase I clinical trial

Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-l-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase adsorbtion and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m², achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 μM for the “therapeutic window” of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 ± 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 ± 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites. Received: 21 July 1996 / Accepted: 20 January 1997     

Establishment of lymphomatous cell lines from bone marrow samples from patients with Burkitt's lymphoma

A total of 233 bone marrow aspirates were obtained from 43 patients with Burkitt's lymphoma (BL). Lymphoma cells were absent and lymphoblastoid cell lines could not be established from 197 samples, which were characterized by limited initial cell proliferation and development of an adherent population, followed by cell death after 2-4 weeks. In 14 aspirates, after a similar pattern of growth, cell proliferation began again after about 6 weeks, with a rapid appearance and growth of cell clumps from the feeder layer--a type of growth typical of spontaneous lymphoblastoid cell lines. In 22 aspirates, growth of malignant cells was observed in culture and cytocentrifuged, stained smears, including marrow samples from 9 patients in whom the presence of BL cells had not been ascertained or even suspected by cytology. Karyotypic anomalies characteristic of BL were found in these cells: t(8;14) in the majority, two t(8;22), two t(2;8), and one t(2;8;9).     

4-Hydroperoxycyclophosphamide purging of breast cancer from the mononuclear cell fraction of bone marrow in patients receiving high-dose chemotherapy and autologous marrow support: a phase I trial

We designed an ex vivo bone marrow treatment for breast cancer patients receiving high-dose chemotherapy and autologous bone marrow support (ABMS), using 4-hydroperoxycyclophosphamide (4-HC), an active derivative of cyclophosphamide with known activity against breast cancer. This phase I bone marrow purging trial used ficoll-separated mononuclear cells (MNC) (devoid of granulocytes and RBCs), as opposed to the buffy coat. Twenty-five patients with metastatic breast cancer were studied. Patients received three cycles of the Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), fluorouracil, and methotrexate (Duke AFM) regimen, followed by marrow harvest. An MNC fraction of marrow was prepared and treated with 4-HC in concentrations of 20 micrograms/mL (four patients), 40 micrograms/mL (four patients), 60 micrograms/mL (nine patients), or 80 micrograms/mL (eight patients) and cryopreserved. Patients then received high-dose systemic cyclophosphamide, cisplatin, and carmustine, followed by infusion of the purged marrow. The study end point was marrow engraftment, defined as WBC count greater than 1,000 cells per microliter. At the first three dose levels (20, 40, and 60 micrograms/mL 4-HC), there was no significant delay in time to engraftment (19, 20, and 23 days, respectively) compared with the unpurged historical controls (17 days). At 80 micrograms/mL, engraftment was significantly delayed compared with the lower concentrations (P = .027), and further escalation of 4-HC was not attempted. A significant correlation was observed between the time of leukocyte engraftment and the 4-HC concentration (P = .017). With a methylcellulose-based tissue culture assay, we demonstrated a statistically significant correlation between the colony-forming unit-granulocyte-macrophage (CFU-GM) content in the purged marrow and the days to engraftment. Ninety-five percent of patients responded clinically to the entire program, 55% of them completely. Longer follow-up is required to assess the ultimate benefit of intensive therapy on long-term survival.     

Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer.

PURPOSE: Virus-directed enzyme prodrug therapy depends on selective delivery of virus encoding a prodrug-activating enzyme to tumor, followed by systemic treatment with prodrug to achieve high levels of the activated cytotoxic at the intended site of action. The use of the bacterial enzyme nitroreductase to activate CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a short lived, highly toxic DNA cross-linking agent has been demonstrated in tumor xenografts. In this study, we report the first clinical trial investigating the feasibility, safety, and transgene expression of a replication-defective adenovirus encoding nitroreductase (CTL102) in patients with liver tumors. PATIENTS AND METHODS: Patients with resectable primary or secondary (colorectal) liver cancer received a single dose of CTL102 delivered by direct intratumoral inoculation 3 to 8 days before surgical resection. RESULTS: Eighteen patients were treated with escalating doses of CTL102 (range, 10(8)-5 x 10(11) virus particles). The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response. Dose-related increases in tumoral nitroreductase expression measured by immunohistochemical analysis have been observed. CONCLUSION: Direct intratumoral inoculation of CTL102 to patients with primary and secondary liver cancer is feasible and well tolerated. The high level of nitroreductase expression observed at 1 to 5 x 10(11) virus particles mandates further studies in patients with inoperable tumors who will receive CTL102 and CB1954.     

Myeloablative therapy and unpurged autologous bone marrow transplantation for poor-prognosis neuroblastoma: report of 34 cases

From October 1984 to November 1987, 34 patients aged from 1 year 1 month to 7 years 7 months with resistant or relapsed neuroblastoma (NB) (group 1, 10 patients), unselected disseminated NB (group 2, 14 patients), or selected disseminated NB (group 3, 10 patients) received myeloablative therapy (MAT) followed by unpurged autologous bone marrow transplantation (ABMT) at the end of an intensive protocol, which included high-dose chemotherapy and surgery to the primary tumor. Median time from diagnosis to MAT and ABMT was 6 months (5 months from last relapse to MAT and ABMT in the relapsed patients). The MAT regimen included vincristine, fractionated total body irradiation (TBI), and melphalan. Seventeen patients were grafted in complete remission (CR), five in very good partial remission (VGPR), 10 in partial remission (PR), and two in progressive disease (PD). The acute toxic death rate was 2.9%. The overall progression-free survival was 29%. The median progression-free survival was 20 months for the 17 patients grafted in CR, 6 months for the five patients grafted in VGPR, and 12 months for the 10 patients grafted in PR.     

益康唑体外净化后自体骨髓移植治疗白血病小鼠的实验研究

目的 建立一个小鼠急性髓系白血病(AML)净化后骨髓移植的模型并探讨益康唑(Econazole,Ec)药物净化的效果.方法 利用小鼠P815 AML细胞转染新霉素抗性基因,即NeoR基因后与小鼠骨髓细胞混合,体外短期培养净化;通过移植后血液系统恢复,白血病微小残留病变和小鼠的存活进行评估.结果 接受放射治疗后经尾静脉注射P815/NeoR细胞而未经净化的小鼠100%可检测到白血病细胞;Ec体外净化后骨髓移植小鼠造血恢复没有延迟,而残留白血病细胞阳性率明显降低且小鼠的总生存明显改善.结论 实验所设计的P815/NeoR小鼠AML净化后骨髓移植模型简单易行、重复性好;Ec可用于AML的净化后骨髓移植.     

骨髓涂片和流式细胞术在神经母细胞瘤骨髓转移微小病灶检测中的临床应用

目的:探讨骨髓涂片和流式细胞术在神经母细胞瘤骨髓转移微小病灶检测中的临床应用价值.方法:回顾性分析2019年01月至2020年10月我院收治的经病理确诊为神经母细胞瘤患者126例,收集患者临床资料,特别是在治疗期间同时同部位进行骨髓穿刺行骨髓涂片和流式细胞术(flow cytometry,FCM)检测,分析两种方法在神...     

Suitability of a new stable acetal analogue of aldoifosphamide for purging leukemic cells from human bone marrow

The in vitro cytotoxic properties of acetaldoifosphamide, a new chemically stable bis-acetate analogue of aldoifosphamide that requires enzymatic activation by cellular carboxylate esterases, has been compared with that of 4-hydroperoxycyclophosphamide (4-HC). On a molar basis, acetaldoifosphamide was 8-10 times more potent than 4-HC against two different human leukemic myeloid cell lines, but only twice as potent as 4-HC against normal bone marrow granulocyte-macrophage colony-forming cells (GM-CFC). Acetaldoifosphamide retained its activity against leukemic cell lines that were highly resistant to the antileukemic drugs doxorubicin and m-AMSA. GM-CFC doubling times after exposure of bone marrow to high concentrations of acetaldoifosphamide in suspension cultures were 6-12 hours. Similar doubling times were obtained after incubation of marrow with 4-HC. Acetaldoifosphamide has a sparing effect on hematopoietic stem cells that is similar to that found for 4-HC; however, it is considerably more potent than 4-HC. Acetaldoifosphamide is different from 4-HC in its chemical stability and its unique requirement for carboxylate esterase activation. We conclude that acetaldoifosphamide may have advantages over 4-HC for in vitro purging of leukemic cells from human bone marrow.     

In vitro synergism of 4-hydroperoxycyclophosphamide and cisplatin: relevance for bone marrow purging

Summary Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide (4-HC)-purged bone marrow gives long-term remission in almost half of relapsed acute nonlymphocytic leukemia and non-Hodgkin's lymphoma patients, but relapse of disease is the main cause of failure, suggesting ineffective purging in some cases. Cisplatin (CP) has activity against a variety of human tumors and is not commonly used for initial therapy of leukemia and lymphoma. Using established human leukemia cell lines, combinations of 4-HC and CP were investigated as a potential regimen for improving the ex vivo removal of leukemia cells from bone marrow. The cell lines (K-562 and Raji) were incubated for 1 (4-HC) or 4 h (CP), washed, and assayed for inhibition of colony formation in semisolid media. In both cell lines, CP (4 h) was more potent than 4-HC (1 h). Combinations of the drugs in various molar ratios were studied after the cells were sequentially incubated with 4-HC and CP. The effects of the drugs were analyzed using the multiple drug-effect analysis of Chou and Talalay [6]. Analysis of data on in vitro inhibition of colony formation suggested that all combinations studied were synergistic in both cell lines, with the greatest synergism being found in the Raji cell line. In addition, for K-562 cells we could detect at least a 4.6 log reduction in cloning with the CP:4-HC combination (1:10 molar ratio). We conclude that CP is a potential candidate in drug combinations for ex vivo bone marrow purging because of its high potency against human leukemia cell lines, its synergistic activity in combination with 4-HC, and its ability to reduce a high tumor burden when combined with 4-HC.Supported in part by grants from the Oliver S. and Jennie R. Donaldson Charitable Trust and from the Westvaco Corporation. Portions of this work were presented at the 78th Annual Meeting of the American Association for Cancer Research, Atlanta, Ga, May 20–23, 1987