A second potential hazard is the infusion EBV transformed B cells which have 
been cocultured with the CTL during generation of the T cell lines. This is 
unlikely to constitute an additional risk to the recipient for several reasons. 
First the lymphoblastoid cells are not viable because they have been irradiated 
with 4000cGy and cocultured with known effectors. Second the 
lymphoblastoid cells are donor type and will be spontaneous transformants; 
the recipient has already received unirradiated donor B cells containing the 
same strain of latent virus with the marrow graft. Thirdly we will add 
Acyclovir to the T cell line cultures (Appendix 1) so no productive virus will 
be present in cultures. To confirm that no productive virus is present the 
ability to transform cord blood will be tested. Finally we will monitor levels 
of EBV DNA in peripheral blood by PCR pre and during CTL infusions. 
3.9 Availability of Gene Marking 
In June 1991, St. Jude Children's Research Hospital was the first center 
outside the NIH to receive RAC/FDA approval for human gene transfer (26- 
28). Amongst the protocols approved was the insertion of a selectable marker 
gene (neomycin resistance) in the LNL6 vector into the marrow of children 
with AML in remission, neuroblastoma in remission or advanced 
neuroblastoma. This marrow is reinfused as an autologous bone marrow 
transplant (ABMT). The primary aim is to determine whether AML or 
neuroblastoma cells at relapse are marked, since 50-70% of the children will 
likely relapse after treatment on these frontline studies. As of November 
1992, sixteen patients have received gene marked marrow. Two patients with 
AML have relapsed and in both cases the neomycin resistance gene has been 
detected in leukemic cells at the time of relapse showing that leukemic cells 
in so called remission marrow can contribute to relapse (29). In addition the 
study has provided information about hemopoietic reconstitution and 
expression of leukemic vectors as the neomycin resistance gene has been 
detected in hemopoietic cells and T lymphocytes for up to a year post 
autograft (30). 
The expertise in the BMT laboratory in this area thus affords an opportunity 
to monitor the survival and recirculation of EBV specific cytotoxic T 
lymphocytes administered to BMT recipients. Should the patient develop 
GVHD, we will analyze part of the routine tissue biopsy by PCR to determine 
if gene marked T cells are present. 
STUDY DESIGN 
4.1 EBV Lymphoblastoid Cell Line Preparation 
At the time of final donor-recipient compatibility typing by MLC, 5cc extra 
blood will be requested through Dr Turner's laboratory and given to Dr 
Recombinant DNA Research, Volume 17 
