A novel potent Fas agonist for selective depletion of tumor cells in hematopoietic transplants

There remains a clear need for effective tumor cell purging in autologous stem cell transplantation (ASCT) where residual malignant cells within the autograft contribute to disease relapse. Here we propose the use of a novel Fas agonist with potent pro-apoptotic activity, termed MegaFasL, as an effective ex-vivo purging agent. MegaFasL selectively kills hematological cancer cells from lymphomas and leukemias and prevents tumor development at concentrations that do not reduce the functional capacity of human hematopoietic stem/progenitor cells both in in vitro and in in vivo transplantation models. These findings highlight the potential use of MegaFasL as an ex-vivo purging agent in ASCT.     

The Use of Archival Bone Marrow Specimens in Detecting B-Cell Non-Hodgkin's Lymphomas Using Polymerase Chain Reaction Methods

The detection of B-cell non-Hodgkin's lymphoma (B-NHL) involving the bone marrow (BM) can be enhanced by assessing immunoglobulin heavy chain (IgH/JH) gene rearrangement using PCR. While the fresh BM aspirate has been the most commonly used specimen, the utility of archival BM tissues has not been extensively evaluated. We studied the BM from 13 patients with nodal B-NHL (7 low-grade and 6 intermediate grade), which were categorized into three groups based on the histologic finding of lymphoma (H) and the presence of a monoclonal IgH/JH band by PCR using fresh BM aspirates (M): (1) H(+)/M(+), 4 cases; (2) H(+)/M(-), 4 cases; and (3) H(equivocal)/M(-), 5 cases. Archival tissues available for study included paraffin-embedded trephine biopsy (TB)/aspirate clots (AC) and air-dried aspirate smears (AS). All TB (13/13) and a subset of AC (5/13) were B5-fixed, and all these tissues failed to yield analyzable DNA. In contrast, sufficient DNA was consistently obtained in AC that were formalin-fixed (8/13). Of these 8 cases, 2/3 of group 1, 3/3 of group 2, and 0/2 of group 3 had a monoclonal IgH band. Using DNA extracted from microdissected lymphoid aggregates morphologically evident in the AC sections, additional positive cases were identified: 1/3 of group 1 and 2/2 of group 3. In those 5 cases that did not have formalin-fixed TB/AC, sufficient DNA was extracted from AS in all cases; one additional positive case was identified in group 1. Overall, 4/4 (100%) of group 1, 3/4 (75%) of group 2, and 2/5 (40%) of group 3 showed molecular evidence of lymphoma. To conclude, archival BM specimens are a useful source of DNA for molecular detection of B-NHL involvement, and formalin appears to be a better fixative than B5. The use of these samples may improve the overall detection sensitivity.     

Newer Options for Treating Drug-Resistant (MDR+) Cancer Cells using Photoradiation Therapy

Emergence of drug resistance with conventional cytotoxic therapy is a major challenge towards the curability of many cancers, especially in patients undergoing autologous BMT with ex-vivo purged hematopoietic support. We have explored the potential role of photoradiation therapy in purging hematopoietic stem cells of various hematological malignancies. Benzoporphyrin derivative, monoacid ring A (BPD-MA), dihematoporphyrin ether (DHE), and MC-540 were evaluated for the “ex-vivo” purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large cell lymphoma cell lines and colony forming-unit leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments four log elimination of tumor cell lines was observed after 1 hr of incubation with BPD-MA or DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA or DHE, the mean recovery of normal BM progenitors was 4-5.2% for granulocyte-macrophage colony forming unit (CFU-GM) and 5-9.8% for burst forming unit erythroid (BFUE).

The T lymphoblastic leukemia cell line CEM and its vinblastine (VBL)-resistant subline CEM/VBL₁₀₀. along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. Our results demonstrated the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation would be useful for “ex-vivo” purging of malignant cells. Other methods to deal with decreasing drug resistance are also detailed.     

High-dose therapy with autologous haematopoietic support in patients with transformed follicular lymphoma: A study of 27 patients from a single centre

Background: The prognosis of patients with transformed follicular lymphoma (FL-t) is poor. The use of high-dose therapy (HDT) with autologous haematopoietic support was therefore evaluated as consolidation of remission.Patients and methods: Twenty-seven patients received high-dose cyclophosphamide and total body irradiation (cyclo + TBI) with autologous bone marrow (BM; n = 24) or peripheral blood progenitor cell support (PBPC; n = 3). BM was treated in vitro with anti-B cell antibodies and complement. Nineteen of 27 patients were treated in first stable remission following transformation. Eight other patients with a history of transformation were treated following a subsequent recurrence of follicular lymphoma (FL).Results: With a median follow-up of 2.4 years, 14 of 27 patients remain alive and in remission; five are alive and free of disease at more than four years. The median survival is 8.5 years. There were two early treatment-related deaths of respiratory failure, and two late deaths of myelodysplastic syndrome (MDS) in remission of lymphoma at 2.8 and 8.5 years.Seven of nine patients having had a recurrence underwent re-biopsy. In two, histology revealed FL, in five, transformed follicular lymphoma. One of the patients with recurrent FL is alive without further therapy, and two of five patients with recurrent FL-t are alive and in remission after further chemotherapy.Conclusions: It is appropriate to consider HDT for younger patients with FL-t in remission. Repeat biopsy should be considered for patients with recurrent disease. There is a risk of late MDS in patients undergoing this treatment.     

The role of quantitative polymerase chain reaction in the management of follicular lymphoma patients

AIMS AND BACKGROUND: In order to increase the prognostic significance of polymerase chain reaction (PCR) data it has been suggested that quantitative PCR can be used to measure tumor burden. However, this option has not yet been definitely supported or refuted in patients with follicular lymphoma (FL). We decided to evaluate whether knowledge of the quantitative level of minimal residual disease and its variations can be of use in the management of FL patients. METHODS: We used qualitative and competitive PCR to study 11 patients with refractory or relapsed FL harboring the t(14;18) translocation who underwent autologous (nine patients) or allogeneic (two patients) stem cell transplantation (SCT). Competitive PCR was performed with a multiple competitor carrying specific sequences including Bcl2/IgH MBR and mcr, and the beta-globin gene. RESULTS: After a median post-SCT follow-up of 44 months (range, 12-62), overall survival was 91% and disease-free survival 82%. The quantitative PCR data showed that: 1) effective chemotherapy before SCT substantially (1-2 log) reduced the tumor burden in the bone marrow (BM); 2) the increase in rearranged DNA detected in BM was associated with disease progression and relapse; 3) a PCR-negative autograft seemed to lead to lasting molecular remission even when it was performed in patients with a low level of BM infiltration before transplant; and 4) allo-SCT made and maintained the BM PCR negative even in the presence of a greater tumor burden before SCT. Six of the nine patients having CR after SCT (four auto and two allo) are in continuous molecular remission. CONCLUSIONS: In FL patients qualitative and quantitative PCR may provide data that can be helpful for the prognostic evaluation of tumor progression and the early detection of impending relapse by highlighting biological features such as the quality of the infused material, the tumor burden at transplant, and the behavior of tumor cells after transplant.     

Usefulness of Lymphokine-activated Killer Cells Generated from Bone Marrow Mononuclear Cells for the Purging of Residual Tumor Cells in Peripheral Blood Stem Cell Graft

Lymphokine-activated killer (LAK) cells were generated from bone marrow mononuclear cells (BM), and the usefulness of the BM-LAK for purging of residual tumor cells in autologous peripheral blood stem cell (PBSC) graft was determined. The BM and peripheral blood lymphocytes (PBL) were obtained from the same bone marrow donors. The BM-LAK and PBL-LAK were generated by incubation with interleukin-2 for 7 days. The BM-LAK demonstrated higher killer activity against a lymphoma cell line Raji than the PBL-LAK. The BM-LAK also had a higher percentage of CD4^-CD8^-CD16⁺ cells than the PBL-LAK, which suggests that their high killer activity is related to these cells. The BM-LAK did not show any killer activity against the PBSC graft. However, they killed tumor cells which contaminated the PBSC graft, and in particular, killed chimeric ber/abI messenger RNA-positive residual leukemic cells. These results suggest that the BM-LAK may be applicable for purging. As the BM-LAK possess higher killer activity than the PBL-LAK, they may be more useful than the PBL-LAK.     

Comparative activities of rabbit complements of different ages using an in-vitro marrow purging model

In order to determine the best time to obtain complement for use in marrow purging in vitro, we have used 51Cr-release and limiting dilution assays (LDA) to evaluate the ability of serum from rabbits 20-30 (25d), 30-40 (35d) and 40-50 (45d) days old to lyse a series of neoplastic target cells in the presence of complement activating antibodies. Utilizing a limiting dilution assay (LDA) to measure log depletion of tumor cells in a 20-fold excess of normal bone marrow, treatment with monoclonal antibody and 25d complement depleted at least 4 logs of leukemia or lymphoma cells. 35d and 45d complements were approximately one log less effective. When normal bone marrow alone was treated with either J5 (CD10) antibody alone, each complement alone or a combination of J5 antibody with each complement, there was no significant depletion of hematopoietic progenitors in any subgroup. These results suggest that complement from 20-30 day old rabbits should be used for the purpose of ex-vivo purging due to its highly sensitive and specific activity.     

Removal of cells from a malignant B-cell line from bone marrow with immunomagnetic beads and with complement and immunoglobulin switch variant mediated cytolysis

In this report we describe the generation of complement (C′) fixing IgG2b CD19 and CD22 monoclonal antibodies (mAbs) by the isolation of immunoglobulin (Ig) class switch variants using a simple and efficient method for the selection of spontaneously mutating hybridomas. The aim of this study was to compare the efficacy of C′-mediated cytolysis vs immunomagnetic (IB) depletion of tumor cells from mixtures of malignant B cells and normal bone marrow. In a clonogenic assay employing the B-cell lines Namalwa and OCI.LY1, we found that the use of immunomagnetic beads warranted a highly efficient tumor cell removal independent of the Ig isotype of the mAbs used. Elimination of up to 4 log was achieved using a cocktail consisting of CD19, CD20, CD22 and CD37 B-cell mAbs. A less efficient killing of 2 log was obtained by C′ lysis using IgM mAbs, while only about 1 log tumor cell elimination was obtained using IgG2b mAbs. Immunomagnetic purging, besides being more effective than C′-mediated cytolysis, is easier to handle and more rapid.     

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support

Summary Detection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identity of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.     

Differential effects of cladribine and gemcitabine on erythroid and granulocytic progenitors from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal neoplastic disease that originates in a pluripotent stem cell. Selection of normal progenitors by graft-purging may improve the outcome after autologous transplantation. In our methylcellulose assays, the nucleoside analogs cladribine (2-CdA) and gemcitabine (dFdC) showed more prominent inhibitory effects on CML than normal bone marrow (BM) progenitors. For dFdC, however, long-term incubations were necessary to achieve complete inhibition. Deoxycytidine kinase, the key enzyme of both 2-CdA and dFdC metabolisms, was only partially responsible for this differential sensitivity. We suggest that 2-CdA and dFdC might be helpful in purging of CML BM cells before autologous BM transplantation. Further studies on more primitive cells are warranted.     

Reactivity of anti-CD15 monoclonal antibody PM-81 with breast cancer and elimination of breast cancer cells from human bone marrow by PM-81 and immunomagnetic beads.

The CD15 carbohydrate antigen, lacto-N-fucopentaose III is expressed on a variety of human cancer cells including acute myeloid leukemia, small cell carcinoma of the lung, and colorectal carcinomas. We have found that cells from breast cancer cell lines and patient-derived tissue are strongly CD15 positive, as seen by binding to the PM-81 monoclonal antibody. In this report we show that monoclonal antibody PM-81 and immunomagnetic beads can remove breast cancer cells from bone marrow and thus be used as "purging" agents for autologous bone marrow transplantation. PM-81 and immunomagnetic beads removed up to 3 log of SK-BR-3 and MCF7 breast carcinoma cell line cells while minimally affecting normal hematopoietic progenitor cells. This technique may be useful for purging marrow for autologous bone marrow transplantation in breast cancer.     

Quantitative real-time polymerase chain reaction for detection of circulating cells with t(14;18) in volunteer blood donors and patients with follicular lymphoma

The chromosomal rearrangement t(14;18)(q32;21) involves the major (MBR) or minor (mcr) breakpoint cluster regions and the immunoglobulin heavy chain joining regions (JH) in most follicular lymphomas. As a first step towards determining the clinical significance of circulating cells with t(14;18), we detected and quantitated circulating cells in samples obtained from volunteer blood donors and follicular lymphoma patients. The t(14;18) was co-amplified with beta-actin with real-time quantitative PCR (QRT-PCR) in reactions containing 1 microg of DNA from peripheral blood or bone marrow aspirates. The cell number was quantitated using linear regression and an external standard of serially diluted DNA from cell lines with MBR/JH or mcr/JH rearrangements. At dilutions of 10(5) and 106, sensitivity was 100 and 55% for MBR/JH, and 100 and 10% for mcr/JH rearrangements. Among 102 volunteer blood donors MBR/JH vs. mcr/JH amplicons were detected in 22 vs. 4% with duplicate 1 microg DNA reactions, and in 41 vs. 6% with a total 10 microg DNA analyzed in multiple reactions. Among volunteer blood donors the mean number of circulating cells with MBR/JH vs. mcr/JH rearrangements were 0.8 vs. 0.1/microg DNA, and exceeded the upper normal limit (defined as the mean of all volunteer samples plus two standard deviations) in 3% vs. 2%, respectively. Analysis for MBR/JH rearrangements revealed that follicular lymphoma patients vs. volunteer blood donors were positive in 76% vs. 22% (p = 0.008 by Fisher's exact test); that the mean number of MBR/JH cells per microg of DNA was 91 vs. 0.8 (p = 0.0002 by Mann-Whitney test); and the number of the MBR/JH cells exceeded the upper normal limit in 32% vs. 3% of subjects (p = 0.0001 by Fisher's exact test). Circulating cells with mcr/JH were not detected among any of these 25 lymphoma patients. We conclude that patients with follicular lymphoma are more frequently positive, have higher numbers of circulating cells with t(14;18), which exceed upper normal limit more frequently than in volunteer blood donors.     

Bone Marrow Purging in Acute Leukemia with Alkyl-Lysophospholipids: A New Family of Anticancer Drugs

The alkyl-lysophospholipids are a new family of anticancer drugs which target the cell membrane as their site of action. Enzymes involved in signal transduction (protein kinase C and phosphatidylinositol phospholipase C), phospholipid biosynthesis (lysophosphatidyl acyltrans-ferase and CTPcholinephosphate cytidylyltransferase) and maintenance of membrane integ rity (Na, K ATPase sodium pump) are inhibited.

A unique feature of the alkyl-lysophospholipids is their selective cytotoxicity to neoplastic cells. This suggests that the compound would be an excellent agent for purging residual leu-kemic cells from marrows of patients in remission prior to autologous bone marrow trans plantation. Preclinical studies in a murine leukemia model and in an in vitro human system demonstrated successful elimination of leukemic cells from a mixture of normal and leukemic marrows. Twenty-nine poor risk patients with acute leukemia underwent autologous bone marrow transplantation and were reinfused with marrow treated in vitro with edelfosine. Nine of these patients remain in remission free of leukemia from 368 to 1369 days. These en couraging results warrant further investigation.     

Indication for bone-marrow harvesting and purging in Burkitt's Lymphoma: a three-year experience

Between 1980 and 1983, 317 bone-marrow (BM) aspirates from 63 cases of Burkitt's lymphoma (BL) were studied with an in-vitro liquid culture monitoring system. BL cell lines were obtained in culture from 14 of 15 patients with cytologically positive marrow. The in-vitro monitoring system was shown to be useful regardless of Epstein-Barr virus (EBV) status, clinical status of patients and cytogenetic anomalies (8 t(8;14) 2 t(8;22) 2 When BM was cytologically normal or suspect, i.e., less than 5% BL cells, the in-vitro monitoring system was shown to be more sensitive than cytological examination in 25 of 56 cases (44%). The sensitivity of the test is 1/100 000, i.e., 3 logs inferior to the level of detection by cytology. When BM was harvested from all patients in complete remission after two months of chemotherapy, marrow purging proved not to be necessary (38/38 negative cultures); 7/10 of those patients will have been harvested to no purpose (no more than 30% with indication for autologous BM transplantation). When BM was harvested at relapse or in partial remission, the purging procedure was shown to be necessary in 9 of 16 cases (56%).     

Entourage: the immune microenvironment following follicular lymphoma

In follicular lymphoma, nonmalignant immune cells are important. Follicular lymphoma depends on CD4+ cells, but CD8+ cells counteract it. We hypothesized that the presence of follicular lymphoma is associated with higher CD4+ than CD8+ cell numbers in the tumor microenvironment but not in the immune system. Using flow cytometry, pre-treatment and follow-up CD4/CD8 ratios were estimated in the bone marrow, blood and lymph nodes of untreated follicular lymphoma patients in two independent data sets (N(1)=121; N(2)=166). The ratios were analyzed for their relation with bone marrow lymphoma involvement. Bone marrows were also investigated with immunohistochemistry. In either data set, the bone marrow CD4/CD8 ratios were higher in bone marrows involved with lymphoma (P=0.043 and 0.0002, respectively). The mean CD4/CD8 ratio was 1.0 in uninvolved and 1.4 in involved bone marrows. Also higher in involved bone marrows were CD4/CD56 and CD3CD25/CD3 ratios. No blood or lymph node ratios differed between bone marrow-negative and -positive patients. Sequential samples showed increased bone marrow CD4/CD8 ratios in all cases of progression to bone marrow involvement. Immunohistochemistry showed CD4+, CD57+, programmed death-1+, forkhead box protein 3+ and CD21+ cells accumulated inside the lymphoma infiltrates, whereas CD8+, CD56+ and CD68+ cells were outside the infiltrates. This study provides evidence in vivo that the microenvironment changes upon follicular lymphoma involvement.     

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。     

Minimal residual disease (MRD) in non‐Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real‐time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST?/RQ? by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined “borderline” (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST?/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ?. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next‐generation sequencing have the potential to overcome the current limitations.     

Granulocyte-Macrophage Colony Stimulating Factor in Autologous Bone Marrow Transplantation: Augmentation of Graft versus Tumor Effect via Antibody Dependant Cellular Cytotoxicity

Several immunomodulatory approaches have been explored with the aim of inducing a graft versus tumor effect (GVT) in autologous bone marrow transplantation (ABMT). Granulocyte-macrophage colony stimulation factor (GM-CSF) has been reported to induce antibody dependent cellular cytotoxicity (ADCC) via stimulation of peripheral blood neutrophils, lymphocytes, and monocytes. We investigated the role of GM-CSF in inducing ADCC via bone marrow (BM) macrophages against murine and human tumor cells both in vitro and in vivo.

Our data shows that stimulation of murine BM macrophages with GM-CSF induced a potent ADCC against a murine melanoma in vitro. Treatment of tumor bearing mice with a combination of GM-CSF and antibody against melanoma resulted in a significant reduction in the dissemination of melanoma both in a nontransplant as well as in a transplantation setting. Adoptive transfer of BM macrophages obtained from animals undergoing treatment with GM-CSF plus antibody significantly reduced the spread of tumor in secondary recipients; this effect was seen only in mice undergoing bone marrow transplantation. GM-CSF treatment of human BM macrophages induced a significant ADCC against a human melanoma and a lymphoma in vitro, as well as against a human lymphoma implanted in nude mice. Treatment with GM-CSF alone or with antibody alone was ineffective in controlling the dissemination of tumors both in transplantation as well as in nontransplant situations. These observations indicate that treatment with GM-CSF plus tumor specific monoclonal antibodies after ABMT may induce a GVT effect and bring about the eradication of residual disease.     

Graft purging in autologous bone marrow transplantation: a promise not quite fulfilled

Clonogenic tumor cells contained within hematopoietic stem cell (HPC) grafts may contribute to relapse following autologous transplantation. Graft purging involves either in vivo or ex vivo HPC manipulation in order to reduce the level of tumor cell contamination. Some phase II trials suggest that patients who receive purged products may have a superior transplant outcome. Phase I trials demonstrate the feasibility of purging methods including ex vivo graft incubation with chemotherapeutic drugs, monoclonal antibodies and complement, and CD34+ cell selection. A phase II trial in follicular non-Hodgkin's lymphoma demonstrates that patients who receive HPC products purged negative for bcl-2 gene rearrangements have a superior outcome, compared with patients who receive polymerase chain reaction (PCR)-positive products. This finding, however, has not been confirmed in a randomized trial. HPC purging has demonstrated no benefit in a phase III trial in myeloma. Phase II trials in acute myelogenous leukemia show comparable outcomes for patients who receive either purged or unpurged HPC grafts. Limitations of purging include possible progenitor cell loss, delayed engraftment, and qualitative immune defects following transplant. Data to justify routine use of HPC graft purging are insufficient. Phase I and II data support development of phase III trials of both in vivo and in vitro purging methods.     

Bcl-2基因重排在恶性淋巴瘤微小残留病变检测中的应用

目的建立敏感的检测淋巴瘤微小残留病变（ＭＲＤ）的方法,探索Ｂｃｌ２基因重排在恶性淋巴瘤的分期、化疗疗效和预后评估中的意义。方法多聚酶链反应（ＰＣＲ）检测Ｂｃｌ２／ＪＨ基因重排,系列稀释试验检测该方法的敏感性。结果９种恶性淋巴瘤细胞系中,ＳｕＤＨＬ４,ＳｕＤＨＬ６有Ｂｃｌ２／ＪＨ基因重排,系列稀释试验检测该方法的敏感度达１∶１０５。滤泡性淋巴瘤（ＦＮＨＬ）患者１６例中,４例外周血和骨髓中同时检出Ｂｃｌ２（ＭＢＲ）／ＪＨ基因重排,化疗达ＣＲ后仍存在。结论多聚酶链反应检测Ｂｃｌ２／ＪＨ基因重排,是检测滤泡性淋巴瘤患者微小残留病变的一个快速、有效、敏感的方法,对疾病的分期、疗效和预后评估有一定的临床应用价值。