Chronic myelogenous leukemia (CML) is a disease that starts as an indolent non-life-threatening disorder, and evolves into a fulminant acute leukemic transformation following acquisition of somatic mutations which complement the Philadelphia chromosome (Ph) translocation. 1 Alpha-interferon has been shown to delay the onset of the blastic phase of this disease in 25% of individuals, 2 and allogeneic bone marrow transplantation is a curative therapeutic intervention which is applicable in an additional 25% of individuals. 3 Intensive systemic therapy followed by hematopoietic reconstitution with autologous cells has been used in this disease to provide a therapeutic option for individuals with CML who are not eligible for alpha-interferon or allogeneic bone marrow transplantation. This depends on collection of autologous bone marrow are peripheral blood cells early in the recovery from myelosuppression induced by conventional dose chemotherapy, as developed by Carella and his coworkers. 4 We have reproduced his findings in that we have found that diploid preparations of peripheral blood cells can be collected from 25–40% individuals so treated. The results of autologous bone marrow transplants, using autologous cells collected early in the phase of hematopoietic recovery from the myelosuppression induced by conventional dose chemotherapy, has suggested that the relapse that occurs following this procedure arises in part from leukemic cells present in the infused cells. 6 The use of retroviral 66marking to tag the infused cells in the autologous transplant has also suggested that leukemic cells in the transplant are also responsible for relapse. 7 In this review, we will summarize the data that have led to these conclusions.

We conducted a double retroviral vector (RV) gene marking trial to test for the possible contribution to relapse of follicular non-Hodgkin's lymphoma (FNHL) cells present in bone marrow (BM) and peripheral blood (PB) grafts used for hematopoietic reconstitution of patients undergoing myelaoblative chemotherapy and autologous transplant. CD34 positive selection using the CellPro Ceprate CD34 column was performed on PB mononuclear cells obtained after cyclophosphamide/G-CSF mobilization. CD34 positive cells were exposed for 4-6 hours to the LNL6 or G1 Na RV in the absence of growth factors or stromal monolayers. One week later, BM mononuclear cells were similarly processed. Patients then received total body irradiation (TBI), cyclophosphamide, and etoposide followed by infusion of both PB and BM CD34 positive cells. Semiquantitative Southern blot analysis of DNA t(14;18) amplification products showed approximately a three log reduction in t(14;18) positive cells after CD34 positive selection. The first patient showed evidence of engraftment with RV positive BM and PB cells for 9 months. He relapsed one year after transplant. At relapse, one year after transplant, he had lost evidence of RV positive cells in ficolled mononuclear BM and PB cells as well as in CD19 positive cells. The second and third patients showed evidence of engraftment with RV positive cells up to 9 and 6 months post BMT respectively. The second and third patients are still in clinical remission. Our results demonstrate engraftment of RV transduced hematopoietic cells in the PB and BM for up to 9 months.

Most patients relapse after high-dose chemotherapy (HDCT) with autologous stem-cell transplantation (ASCT) for metastatic breast cancer. Further chemotherapy immediately after hematopoietic recovery from ASCT is not given for fear of irreversibly damaging the newly engrafted stem cells. In a pilot chemoprotection trial, autologous CD34+ cells from patients with metastatic breast cancer were exposed to a replication-incompetent retroviral vector carrying MDR-1 cDNA and then reinfused after HDCT. Immediately on recovery, patients received multiple courses of escalating dose paclitaxel. All of the 10 patients tolerated reinfusion of modified cells without any toxicity and had myeloid engraftment within 12 days (range, 11-14). The bone marrow cells of three patients contained vector MDR-1-positive cells only at the time of the first course of posttransplant paclitaxel, indicating that the MDR-1 vector-modified cells had only short-term engrafting potential. A total of 83 courses of paclitaxel were administered starting at a median of 30 (range, 21-32) days from ASCT. The median dose of paclitaxel was 225 mg/m2 and the median interval between paclitaxel cycles of therapy was 21 (range, 20-41) days. Five of the six CR patients were able to receive all of the 12 courses of paclitaxel. Three patients who had achieved less than a complete response to the HDCT (2 patients) and partial response (1 patient) were converted to complete clinical responses during the 12 cycles of paclitaxel. No delayed toxicity or bone marrow failure was noted in these patients with a median follow-up of 2 years from ASCT. This is the first study of chemotherapy immediately after transplantation with autologous CD34+ cells. These data indicate that paclitaxel can be safely administered immediately after ASCT without any delayed toxicities. Paclitaxel given immediately after ASCT can further improve the response to pretransplant chemotherapy in patients with advanced breast cancer.

Genetic modification for cancer treatment has involved the introduction of chemotherapy protection and sensitization genes into normal and tumor cells, respectively, for the purpose improving the outcome of conventional approaches to the treatment of solid tumor neoplasms. This paper will review the use of multidrug resistance‐1 retroviral vectors and cytosine deaminase adenoviral prodrug activation vectors for this purpose.

High-dose chemotherapy with hematopoietic progenitor cell support is administered increasingly to selected categories of patients with high-risk malignancies. Bone marrow and/or peripheral blood progenitor cells (PBPCs) are commonly cryopreserved with the cryoprotectant dimethyl sulfoxide (DMSO), which can cause a variety of systemic side effects when the graft is thawed and infused. The progenitor cells thought to be responsible for hematopoietic recovery express the CD34 antigen and constitute 1% to 3% of the marrow cells and 0.5% of the PBPC fraction. Transplantation of a CD34+ graft would markedly reduce the volume and thus the amount of DMSO required, thereby decreasing the infusion-related toxicities. In this study, 89 high-risk breast cancer patients received high-dose therapy and were randomized to receive an autologous CD34+ marrow graft (Arm A) versus a standard buffy coat fraction (Arm B). After marrow infusion, significant increases in diastolic and systolic blood pressure, as well as significant decreases in heart rate, were documented in Arm B compared to Arm A patients (P < .001). None of the patients in Arm A experienced any clinically serious adverse events associated with the marrow infusion compared to 6% of the Arm B patients. The median time to neutrophil engraftment was 13 days for Arm A and 11 days for Arm B patients (P = .218). The median time to platelet engraftment was 27 days for Arm A and 20 days for Arm B patients (0.051). There were no other significant differences between the two arms of the study with respect to thrombocytopenia-related complications or immune function reconstitution. Additionally, patients on Arm A who received ≥1.2 × 106 CD34+ cells/kg had no delay in platelet recovery (22 days), compared to patients on Arm B, who also received greater than 1.2 × 106 CD34+ cells/kg (20 days) (P = .604). In conclusion, this prospective randomized study demonstrates that breast cancer patients who receive high-dose therapy with autologous CD34+ marrow support have reduced marrow infusion-related toxicity, comparable time to neutrophil engraftment and immune function recovery posttransplant, and for those who receive <1.2 × 106 CD34+ cells/kg, comparable time to platelet engraftment compared to women who receive buffy coat fractions of marrow.

We have developed an avidin-biotin immunoadsorption technique in conjunction with a monoclonal anti-CD34 antibody that is capable of selecting CD34+ progenitor cells from marrow and mobilized peripheral blood. Clinical studies with these CD34+ selected cells have shown that the cells are capable of rapid and durable engraftment. In addition, there is significantly less infusional toxicity to the patient because the volume in which the CD34+ selected cells are contained is much less than that of a typical marrow or apheresis buffy coat. Selection of CD34+ progenitor cells also offers other potential advantages, including T-cell depletion of allografts and tumor cell depletion of autografts. CD34+ selection can also be used to facilitate other manipulations of marrow and peripheral blood, including gene transfection, ex vivo stem cell expansion, tumor purging, and progenitor cell banking. Future graft engineering studies are expected to clarify these relationships and enable refinement of the graft to the point at which GVHD can be minimized, graft survival maximized, and relapse-free survival prolonged.

Human Gene TherapyVol. 7, No. 5 News and CommentsUse of Double Marking with Retroviral Vectors to Determine Rate of Reconstitution of Untreated and Cytokine Expanded CD34+ Selected Marrow Cells in Patients Undergoing Autologous Bone Marrow Transplantation. St. Jude Children's Research Hospital, Memphis, TennesseePRINCIPAL INVESTIGATORS Helen E. Heslop, Malcolm K. Brenner, Robert A. Krance, CO-INVESTIGATORS Laura Bowman, John M. Cunningham, Stacye Richardson, Barbara Alexander, Richard Heideman, James M. Boyett, Deo-Kumar Srivastava, Stephen G. Marcus, Ronald Berenson, Shelly Heimfeld, and Sherri BrownPRINCIPAL INVESTIGATORS Helen E. HeslopSearch for more papers by this author, Malcolm K. BrennerSearch for more papers by this author, Robert A. KranceSearch for more papers by this author, CO-INVESTIGATORS Laura BowmanSearch for more papers by this author, John M. CunninghamSearch for more papers by this author, Stacye RichardsonSearch for more papers by this author, Barbara AlexanderSearch for more papers by this author, Richard HeidemanSearch for more papers by this author, James M. BoyettSearch for more papers by this author, Deo-Kumar SrivastavaSearch for more papers by this author, Stephen G. MarcusSearch for more papers by this author, Ronald BerensonSearch for more papers by this author, Shelly HeimfeldSearch for more papers by this author, and Sherri BrownSearch for more papers by this authorPublished Online:20 Mar 2008https://doi.org/10.1089/hum.1996.7.5-655AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byContributions of Gene Marking to Cell and Gene Therapies Cecilia N. Barese and Cynthia E. Dunbar24 January 2011 | Human Gene Therapy, Vol. 22, No. 6The Continuing Contribution of Gene Marking to Cell and Gene TherapyMolecular Therapy, Vol. 15, No. 4Gene transfer to hematopoietic cells - the clinical experience24 April 2009 | European Journal of Haematology, Vol. 59, No. 2Gene transfer trials in clinical haematology17 September 2018 | Journal of Internal Medicine, Vol. 242Stem Cell‐Based Gene Therapy1 June 1997 | The Oncologist, Vol. 2, No. 3A Novel Herpes Vector for the High-Efficiency Transduction of Normal and Malignant Human Hematopoietic CellsBlood, Vol. 89, No. 1 Volume 7Issue 5Mar 1996 InformationCopyright 1996, Mary Ann Liebert, Inc.To cite this article:PRINCIPAL INVESTIGATORS Helen E. Heslop, Malcolm K. Brenner, Robert A. Krance, CO-INVESTIGATORS Laura Bowman, John M. Cunningham, Stacye Richardson, Barbara Alexander, Richard Heideman, James M. Boyett, Deo-Kumar Srivastava, Stephen G. Marcus, Ronald Berenson, Shelly Heimfeld, and Sherri Brown.Use of Double Marking with Retroviral Vectors to Determine Rate of Reconstitution of Untreated and Cytokine Expanded CD34+ Selected Marrow Cells in Patients Undergoing Autologous Bone Marrow Transplantation. St. Jude Children's Research Hospital, Memphis, Tennessee.Human Gene Therapy.Mar 1996.655-667.http://doi.org/10.1089/hum.1996.7.5-655Published in Volume: 7 Issue 5: March 20, 2008PDF download

A retrovirus containing the multiple drug resistance (MDR-1) cDNA, was used to transduce cultures of CD34 selected human marrow cells, on stromal monolayers in the presence of hematopoietic growth factors IL-3 and IL-6, following collection from patients recently recovered from chemotherapy-induced myelosuppression. In one experiment, these CD34 selected cells were grown in Dexter cultures for 35 days or more following MDR-1 transduction, and then plated in methylcellulose. Polymerase chain reaction (PCR) analysis of colonies picked after 10-14 days of methylcellulose culture, using a set of primers that are specific for the endogenous or the retrovirally transduced MDR-1, showed that the long-term culture initiating cells (LTCICs) were transduced by the MDR-1 virus. Analysis of the colonies from the CD34 selected MDR-1 transduced cells, with a reverse transcription (RT) PCR assay that could distinguish viral MDR-1 mRNA from endogenous MDR-1 mRNA, showed that the viral MDR-1 mRNA levels were much higher than that of the MDR-1 mRNA from the endogenous MDR-1 gene in the transduced CD34 selected cells. Fluorescence activated cell sorting (FACS) analysis of the CD34 selected transduced marrow cells within 48 h after the transduction, using the C219 and UIC2 monoclonal antibodies for p-glycoprotein, showed that the transduction frequency under these conditions varied from 7 to 20%. Rhodamine efflux studies showed that this additional p-glycoprotein was functional and that the frequency of cells with high p-glycoprotein levels was higher in the transduced cells than in the non-transduced cells. The resistance to taxol of the CD34 selected transduced cells, as judged by the plating efficiency of clonogenic progenitor cells derived from these cells by growth in methylcellulose supplemented with taxol was much higher in the transduced cells than in untransduced cells. In order to test the reproducibility of the transduction frequency of the retroviral supernatants from PA317 MDR-1 viral producer cells on CD34 selected cells, the virus produced from 12 different lots of supernatants from the PA317 MDR-1 producer cell line was used to transduce CD34 selected marrow cells from four different patients, and to transduce the peripheral blood cells of two additional patients collected following chemotherapy-induced myelosuppression. The supernatant lots used for these transduction experiments were tested by Microbiological Associates (Rockville, MD, USA), by the Mus dunni co-cultivation and amplification tests in the S+L-assay and found to be negative for replication-competent retrovirus, and later approved for human use by the Food and Drug Administration.(ABSTRACT TRUNCATED AT 400 WORDS)

A large volume culture system was developed for the ex vivo expansion of CD34 positive (+) hematopoietic progenitors, using cell donated by 15 patients receiving high-dose chemotherapy with autologous hematopoietic progenitor cell support (AHPCS). Substantial expansion of myeloid (181-fold) and megakaryocyte (41-fold) progenitors cells was demonstrated, using the conditions that we determined to be optimal: CD34+ progenitors cultured unperturbed for 7 (marrow) or 10 (blood) days in Teflon-coated bags with X-Vivo-10 medium containing 10% autologous plasma, 100 ng/ml, respectively, of recombinant stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The studies demonstrated that (a) CD34 selection was necessary to obtain large, clinically relevant numbers of hematopoietic progenitors, (b) the addition of G-CSF to the baseline regimen of SCF/IL-3/IL-6 significantly enhanced the expansion of myeloid progenitors, (c) the addition of IL-1 to SCF/IL-3/IL-6 did not significantly enhance myeloid progenitor cell expansion, (d) CD34+ G-CSF-mobilized peripheral blood progenitor cells (PBPC) produced higher numbers of myeloid progenitors in culture than CD34+ marrow cells, and (e) long-term tissue culture (LTC) assays demonstrate the preservation of long-term initiating cells in ex vivo culture. The short-term and long-term reconstituting capability of CD34+ PBPC cultured in this system remains to be determined and will be evaluated in a clinical trial where they will be used as the sole source of AHPCS following high-dose therapy.

We collected peripheral blood mononuclear cells and bone marrow cells soon after recovery from conventional-dose chemotherapy-induced myelosuppression and transplanted these cells into advanced chronic myelogenous leukemia (CML) patients after treating these patients with 120 mg/kg cyclophosphamide, 750 mg/m2 VP-16, and 1,020 cGy of total body irradiation (TBI). Of the 10 late chronic-phase patients and the eight accelerated-phase CML patients evaluable posttransplant, 90% and 87%, respectively, remain alive posttransplant, whereas none of the three blast crisis CML patients given this therapy remain alive posttransplant. We measured the percentage of Philadelphia chromosome (Ph)-negative cells in the autologous cells collected after conventional-dose chemotherapy-induced myelosuppression before autologous transplant and in the marrow of these same CML patients after autologous transplantation of these cells into recipients treated with the cyclophosphamide, VP-16, and TBI. A direct correlation (correlation coefficient = 0.91) was observed between the level of Ph+ cells in the transplanted cells and the percentage of Ph+ marrow cells after transplant in 21 patients so transplanted. The data show that the chance of generating cytogenetic remissions post-transplant depends on the percentage of diploid cells in the preparations of autologous cells used for transplant and the stage of disease of the patients at the time of collection of the autologous cells.

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSelection and Transplantation of Hematopoietic Stem and Progenitor CellsKaren Auditore-Hargreaves, Shelly Heimfeld, and Ronald J. BerensonCite this: Bioconjugate Chem. 1994, 5, 4, 287–300Publication Date (Print):July 1, 1994Publication History Published online1 May 2002Published inissue 1 July 1994https://pubs.acs.org/doi/10.1021/bc00028a002https://doi.org/10.1021/bc00028a002research-articleACS PublicationsRequest reuse permissionsArticle Views87Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Sixty-six stage IV breast cancer patients received high dose chemotherapy followed by autologous transplantation of CD34-positive(+) cells obtained from the bone marrow and/or granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood. Grafts were examined for the presence of tumor using conventional histology and immunocytochemical staining. Patients achieved a granulocyte count of 500 x 10(9)/liter 10-12 days posttransplant, with a platelet count of > 20 x 10(9)/liter in 14-15 days. Enrichment of CD34+ cells from the peripheral blood progenitor cell (PBPC) collections resulted in a 1.3 to 4.0 log depletion of breast cancer cells from the graft.