Division of Bone Marrow Transplantation (HEH, DRR, MKB), Department of HematologyOncology, and Department of Virology and Molecular Biology (CMR,), St. Jude Children's Research Hospital, Memphis, TN 38105; the Department of Pediatrics (HEH, MKB), and Department of Pathology (CMR) University of Tennessee, Memphis, Memphis, TN 38163

INTRODUCTION

Hemopoietic progenitor cells have been considered an ideal target for gene therapy of many single gene disorders currently treated by bone marrow transplantation as they are easily obtained and manipulated ex vivo. Furthermore genetic modification of stem cells could be sufficient to repopulate the hemopoietic and lymphoid system of an individual for their entire life. However, the level of gene transfer obtained with current gene transfer techniques as wells as concerns about the expression and regulation of new genes in such cells has delayed the use of gene therapy in most patients with these conditions (Miller, 1992; Anderson, 1992). In contrast gene marking studies allow clinically biological questions to be addressed even with the limited efficiency of gene transfer available with current vectors. In our initial studies in autologous bone marrow transplantation (AMBT) , we used gene marking techniques to determine the source of relapse after ABMT and to learn more about the biology of normal marrow reconstitution. In follow up studies we are evaluating the efficacy of purging and the effect of growth factors on reconstitution. We are also using gene marking to monitor the efficacy of an adoptive transfer approach in patients at high risk of developing EBV Iymphoproliferation following allogeneic bone marrow transplantation.

GENE MARKING STUDIES AFTER AUTOLOGOUS BMT

1. Source of Relapse After Autologous Bone Marrow Transplantation

While autologous bone marrow transplantation appears to produce therapeutic benefit in a number of malignant diseases, including acute myeloid leukemia (Santos et al, 1989; Spitzer et al, 1992; Jones, 1993), relapse remains the major cause of treatment failure. The possibility that reinfused leukemic cells may contribute to relapse has led to extensive evaluation of techniques for purging marrow to eliminate putative residual malignant cells (Gorin et al, 1990; Yeager et al, 1986). However it has been unclear whether purging is necessary .One approach to resolving this issue is to mark the marrow at the time of harvest and then find out if the marker gene is present in malignant cells at the time of a subsequent relapse. The first generation studies using bone marrow of patients with AML and neuroblastoma began in September 1991 and January 1992, respectively and closed in March 1993. Nucleated bone marrow ( > 1.5 x 10 high 8 cells/kg of body weight) was taken from the posterior iliac crest and two thirds were cryopreserved immediately. The remaining third was separated on a Ficoll gradient to produce a mononuclear cell fraction which was transduced with either the LNL6 or the closely related GIN retroviral vector for 6 hours as previously described (Rill et al, 1992). Both these vectors encode the neomycin resistance gene which can subsequently be detected in transduced cells by PCR or because it confers resistance to the neomycin analogue G418. At the time of transplantation, both transduced and unmanipulated marrow cells were thawed and reinfused through each patient's central venous line. To assess the efficiency of gene transfer and expression in marrow progenitor cells both pre- and post-transplantation, we obtained mononuclear cells from peripheral blood or bone marrow, separated them on Ficoll-Hypaque gradients and cultured them in methylcellulose as previously described (Rill et al, 1992). Transduced cells were cultured with or without G4l8. PCR was used to detect the transferred NeoR gene in individual colonies or in bulk populations as previously described (Rill et al, 1992). The PCR-amplified products were analyzed by gel electrophoresis (1.5% agarose) and Southern blotting with a 32p-labeled Neo-specific probe. Semiquantitative PCR was also used, with 25 cycles of amplification corresponding to linear signal strength. In the AML study, four of twelve patients have relapsed. In two, the malignant cells contained the marker gene (Brenner et al, 1993b). The third patient was uninformative as marrow mononuclear cells had low levels of NeoR by PCR but no blast colonies grew in G418 and his blasts did not have a leukemia specific marker so the source of the PCR signal could be determined. The fourth patient is currently being evaluated. To definitively prove that the marker gene is in leukemia cells it is necessary to have a collateral leukemia specific marker so it is possible to demonstrate both this marker and the marker gene in the same cell. For example patient 2 was found to be in relapse on a routine bone marrow at 6 months. This patient had two leukemia specific markers. First her blasts co-expressed CD34 and CD56 a combination not found on normal hemopoietic cells. Second she had a complex t(l :8:21) translocation resulting in generation of a AML1/ETO fusion transcript that could be identified by PCR. We were therefore able to sort blasts expressing CD34 and CD56 and show co-expression in a single clonogenic cell of both a leukemia-specific marker (the AML:ETO fusion protein) and the transferred neomycin gene (Brenner et al, 1993b). Similarly, five neuroblastoma patients have relapsed, and in 4 cases, gene marked neuroblastoma cells were detected (Rill et al, 1994). In these patients, identification of marked neuroblastoma cells was confirmed by detection of co-expression of neuroblastoma-specific antigens (for example GD2) together with the transferred marker gene. Four of the neuroblastoma patients relapsed in their marrow, but the fifth had disease recurrence in an extramedullary site in his liver. Biopsy of this extra-medullary site showed the presence of gene marked neuroblasts. We used a limiting cycle PCR technique to estimate the proportion of resurgent neuroblasts that were marker gene positive in each of the fIrst three patients (Rill et al, 1994). The neomycin resistance gene was present in 0.05% to 1% of the GD2 + CD45- cell population at sites of relapse. Since only 1 % of clonogenic neuroblasts present in the autologous marrow transplant were genetically marked the proportion of marked malignant cells in vivo at the time of relapse was similar to the proportion marked ex vivo and infused with the autologous marrow transplant. These data show that residual tumor cells in marrow have considerable clonogenic potential in vivo and suggest that they make a substantial contribution to resurgent disease. These data show definitively that marrow harvested in apparent clinical remission in two pediatric malignancies may contain residual tumorigenic cells and that these cells can contribute to disease recurrence at both medullary and extra-medullary sites.

Gene Transfer to Normal Cells

Two of the twenty one patients died (of disease progression and sepsis) within 21 days of transplantation and could not be further assessed. In the remaining 19 patients, there were no indications that laboratory manipulation of marrow specimens had adversely affected engraftment. None of the patients has shown evidence of malignant transformation or other complications that could be attributed to the gene transfer process. The presence of the gene in hemopoietic progenitor cells in vivo was confirmed by clonogenic assays that showed gene-marked progenitor cells in 15/19 patients at one month after ABMT. We obtained evidence for the contribution of infused marrow to long-term hemopoietic recovery from the 17 patients who have been followed for 6 months or more after transplantation. By clonogenic assay, G418-resistant progenitor cells persisted for six months in 16/17 patients, for one year in 11/11 , for 18 months in 6/6 and at 24 months in the two patients evaluable thus far. Multilineage GEMM colonies became apparent 6 months after transplantation in 3/9 patients (Brenner et al, 1993a). The marker gene was also present and expressed long term in the mature progeny of marrow precursor cells, including peripheral blood T and B cells and neutrophils. The level of transfer varied and was highest in marrow clonogenic hemopoietic progenitors where the percentage of G418 resistant myeloid colonies ranged from 0-29% .In peripheral blood cells expression was variable between the different lineages and was higher by an order of magnitude in myeloid cells than in T lymphocytes. The lowest level of expression was seen in B lymphocytes. These levels of transfer are higher than predicted from animal models and may be attributed to the fact that marrow was harvested during regeneration after intensive chemotherapy, when a higher than normal proportion of stem cells are in cycle. These data also support a substantial contribution to marrow reconstitution from autologous transplants and suggest that this contribution includes long-lived multipotent stem cells. It should be possible to use the genetic marker technique we describe to compare the regenerative potential of marrow versus peripheral blood-derived progenitor cells and hence discover their relative value for hemopoietic rescue.

Purging Studies

We have begun second generation studies of marrow purging using two gene markers to compare marrow purging versus no purging or two different purging techniques. We are using two closely related vectors, G IN and LNL6, which can be discriminated by virtue of differing fragment sizes they produce after PCR amplification. In the AML study 1/3 of the marrow is frozen unpurged as a safety backup. The remaining marrow is split into two aliquots which are marked with GINa or LNL6 and then randomly assigned to purging with 4HC or IL2 (Brenner et al, 1994). At the time of transplant both aliquots are reinfused. If the patient should subsequently relapse detection of either marker will allow us to learn if either of these purging techniques is effective. Eight patients have been treated on this protocol as of November 1994. None have relapsed, but transfer has been seen in normal progenitors albeit at a lower level than in the studies using unpurged marrow. A similar protocol for neuroblastoma will begin accrual imminently (Brenner et al, 1993).

Ex vivo expansion studies

Genetic marking could also be used to determine directly which ex vivo or in vivo combination of cytokines will increase the entry of long-term marrow repopulating cells into cell cycle and thereby reduce the period of marrow hypoplasia and immunodeficiency that follows autologous stem cell transplantation. A number of animal models have suggested that ex vivo treatment with growth factor combinations, with or without the addition of marrow stromal cells or extracellular matrix, can induce stem cells to enter cell cycle and thereby increase both their number and the efficiency with which they can be transduced (Bodine et al, 1989; Moritz et al, 1994). In humans, it certainly appears possible to use growth factors such as IL-l, IL-3 and stem cell factor to increase the numbers of hemopoietic progenitor cells by 10 to 50 fold and to increase the efficiency of gene transfer to levels which may exceed 50% .Unfortunately , it is not certain that such ex viva data will be reflected by results in viva. In primate studies, transplantation of marrow treated ex vivo with the growth factor combinations shown to greatly augment both progenitor numbers and gene transfer rates, have been followed by disappointingly low levels of long term gene expression in vivo. Similarly, in human studies in which SCF, IL- 3 and IL-6 were used to stimulate marrow progenitor cells before reinfusion, expansion of CFUs and a high genetransfer rate ex vivo were associated with in vivo expression that was both low and attenuated (Dunbar et al, 1994). The likeliest explanation for this apparent paradox is that many of the growth factors intended only to induce cycling in marrow stem cells also induce their differentiation and the loss of their self renewal capacity.
Without any proven ex vivo surrogate method for studying the effects of growth factors on stem cell expansion and transducibility , it is possible to use the marker-gene technique to evaluate whether any increase in progenitor cell numbers and transducibility produced by growth factor combinations and cell culture devices ex vivo has an effect in vivo. By using two distinguishable vectors to mark each patient's marrow, we can compare treatment regimens within a patient. Because each patient acts as their own control, it should be possible to discern the effects of any given growth factor regimen on stem cells, even in a cohort as small as 10 patients.

GENE MARKING STUDIES AFTER ALLOGENEIC BMT

Gene Marking of Adoptively Transferred EBV Specific CTLs

We have also begun a marking study in recipients of allogeneic bone marrow obtained from matched unrelated or mismatched family member donors. In these individuals there is greater genetic disparity between donor and recipient and a higher risk of GVHD. In vitro T cell depletion of donor marrow is an effective means of reducing the risk of GVHD but recipients of depleted marrow have delayed immune recovery and an increased incidence of viral infections (Koehler et al, 1994). One such infection is Epstein-Barr virus lymphoproliferative disease (EBV-LPD). EBV usually persists lifelong after primary infection, by a combination of chronic replication in the mucosa and latency in peripheral blood B cells. EBV infected B cells are highly immunogenic and outgrowth only occurs in severely immunocomprimised patients. This complication occurs in 5-30% of patients receiving marrow from mismatched family members or unrelated donors and until recently was almost invariably lethal. Recently several groups have evaluated the effects of infusing donor T cells to treat this complication (Papadopoulos et al, 1994; Servida et al, 1993; Heslop et al, 1994a). Such therapy can induce disease regression, although with a high risk of inducing significant graft versus host disease (Servida et al, 1993; Heslop et al, 1994a). To avoid this latter complication, we are currently administering infusions of EBV specific donor T cells to high risk patients in a Phase I prophylaxis study (Heslop et al, 1994b). These specific T cells are generated by culturing donor T cells with donor derived EBV infected lymphoblastoid cell lines. To determine whether these cells persist and whether they cause adverse effects they are marked with the neomycin resistance gene before administration. As of November 1994 ten patients have been enrolled on this protocol. The level of marking of infused CTL as assessed by semi-quantitative PCR ranges from 0.5-5% , levels which previous experience suggested would be adequate to enable the survival and behavior of these cells to be followed. At the lowest dose level of 107 cells/mē, CTL will comprise 1 in 10,000 mononuclear cells immediately post infusion which is within the detection limits of PCR. The NeoR gene was detected in peripheral blood lymphocytes for up to 18 weeks or more after injection of gene marked cells and in EBV specific CTL lines regenerated form these patients for up to 9 months . We also have evidence that these transferred cells were functional in vivo (Rooney et al, 1994). In two patients, it has been possible to show anti-viral efficacy in vivo with a dramatic fall in EBV DNA following infusion. In an additional patient, both anti-viral and antilymphoproliferative activity were demonstrable. In this individual, cervical, hilar and retroperitoneal lymphadenopathy developed before the CTL were given. Immunohistology was typical of post-transplant lymphoproliferative lymphoma. Administration of CTL was associated with resolution of the lymphadenopathy over a 6 week period; to date there has been no resurgence of his disease (Rooney et al, 1994). It should be possible to use a similar approach to transfer T cells specific for other malignancies with tumor restricted antigens (Brenner et al, 1991).

CONCLUSION

Genetic marking can be used to evaluate the contribution of residual tumorigenic cells in marrow to relapse after autologous bone marrow transplantation. Marker studies should also prove valuable for assessing the efficacy of purging and for learning about the impact of hemopoietic growth factor treatment of harvested marrow or peripheral blood stem cells on subsequent progenitor cell growth and development in vivo. Finally gene marking should help evaluate the toxicity and efficacy of T cell adoptive transfer in recipients of allogeneic BMT .

ACKNOWLEDGEMENTS

This work was supported in part by NIH Cancer Center Support CORE Grant P30 CA21765, CA 61384, CA 20180 and the American Lebanese Syrian Associated Charities (ALSAC). We would like to thank all our clinical and laboratory associates at St Jude Children's Research Hospital who helped make this work possible and Nancy Parnell for word processing.

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