complete disappearance of IGF-I transcripts shown by Northern blot hybridization as well as IGF-I 
protein as determined by immunocytochemistry with antibody to IGF-I and according to techniques 
previously demonstrated for the rat glioblastoma (32,36). 
Preparation of Transfected Tumor Cells for Reinjection To insure quality control 
appropriate for the cell material to be reinjected subcutaneously, the following tests will be done: 
1. 48 hours prior to clinical use, sterile cultures for routine bacteria and mycoplasma and 
endotoxin levels will be obtained and assayed in the bacteriology and virology laboratories 
of University Hospitals. 
2. On the day of harvest of the transfected cells, the cells will be trypsinized and placed into 
50 ml conical tubes (Falcon) for centrifugation. An aliquot of supernatant will be obtained 
3. In order to remove materials from cells prior to reinjection, the transfected cell 
preparations will be washed and pelleted X 3 in 250 ml volumes of phosphate buffered 
saline pH 7.40. Viable cell count will be determined by exclusion of Trypan blue. The final 
preparation of cells will be suspended in preservative free normal saline for injection at a 
concentration of 50 million cells per ml. 
-Patient Procedures, Conventional Treatment, and Time Schedule 
Patient Groups. All patients will have undergone biopsy and/or resection of their 
supratentorial Glioblastoma multiforme as deemed appropriate by their attending neurosurgeon at the 
time of entry into the study. The experimental treatment will not begin until after radiation therapy 
(if prescribed) is complete. Patients will have been weaned from any high potency glucocoricoids to 
the extent possible as dicated by their residual tumor and cerebral edema. The first treatment will 
take place at about 6 weeks after the original surgical intervention. The patients will be consecutively 
divided into three experimental groups consisting of four patients each. Each patient will receive his 
or her own transfected and irradiated cells injected subcutaneously over the left forearm. Four patients 
will receive 10X10^ transfected cells; four, 50X10^; and four, 200X10^. The patient's glioma cells will be 
maintained in continuous culture and additional booster doses will be injected in the opposite forearm 
alternately at one and three months using the same initial dose. At six months assuming acceptable 
toxicity, patients would be entered into a randomized study of treatment efficacy. 
Clinical Observation. Patients will be admitted to the Clinical Research Center (CRC) at 
University Hospitals six to eight weeks following surgery and on the day prior to experimental 
treatment. They will stay in the CRC for the first 48 hours after reinjection of the transfected and 
irradiated tumor cells. In that setting frequent monitoring of vital signs and temperature will be 
performed looking for evidence of systemic effects. Patients will then be discharged to outpatient 
followup. Each will be given a thermometer and asked to record oral temperatures twice daily while 
at home over the six weeks after the injection. Each will be seen on the Clinical Research Center for 
blood work and brief physical examination three times in the first week then weekly for three weeks. 
General Physical Examination A complete medical history and physical examination, 
including detailed neurologic exam and examination of the lymph nodes will be performed on each 
study subject upon signing consent for the trial before surgery, at the start of the trial (after completing 
the usual radiation treatment protocol). Evaluation for toxicity will take place over the first 48 hours 
in the Clinical Research Center and daily for the next week, followed by weekly for a period of three 
weeks. After the second injection, patient will be again monitored for 48 hours in the Clinical Research 
Center. Follow up will then be weekly for three weeks, every tv/* weeks for four months and monuily 
thereafter. 
Recombinant DNA Research, Volume 18 
[137] 
