Protocol ID93-008 
Page 14 
The presence of GlNa transduced cells in tumor tissues and control sites will be detected by 
PCR using neo specific primers as described in Section 2.8 and Appendix 1 utilizing an 
improved PCR and Southern analysis procedure that included 32p specific neoprobe. 
8.1 In successful experiments a significantly increased neo^ signal detection by PCR and 
Southern analysis should be observed in tissues obtained at tumor sites. These 
results will be compared in biopsy material from normal tissues (peritoneum, omentum 
and lymph node tissue). To control for the presence of neo^^ marked cells in blood, 
samples of blood will be obtained concurrently for PCR analysis. (See 10.0 Statistics 
Section for analysis of data). 
8.2 The fold of enrichment of transduced cells at tumor sites will be determined by PCR 
performed on DNA extracted from tumor and nontumor tissue samples. The DNA will 
be extracted by proteinase K/phenol or Quagen (midi) DNA kit for blood and peritoneal 
fluid samples and analyzed by PCR. PCR quantitation will be performed on at least 
three replicates and results compared against the K562 neo^^ standard. Extracted DNA 
samples will also be run in several dilutions. 
8.3 In addition to these objectives we will assess the persistence of neo^ marked cells in the 
abdominal cavity we will obtain samples from the peritoneal cavity (through the 
catheter during the 18 days of rIL-2 administered) and washings obtained at 
laparoscopy. 
9.0 CRITERIA FOR REMOVAL FROM STUDY 
9. 1 Progressive Disease. 
9.2 Intercurrent Illness which prevents further treatment. 
9.3 Grade IV toxicity in the absence of a significant response which in the judgment of the 
investigator does not warrant the risk of further treatment. 
9.4 Decision of the patient to withdraw from the study. 
9.5 General or specific changes in the patient’s condition which would render further rIL-2 
therapy unacceptable treatment in the judgment of the investigator. 
10.0 STATISTICAL CONSIDERATIONS 
A justification is provided for undertaking a pilot study of 9 patients. It will be 
demonstrated that It is virtually certain that some lymphocytes sampled from each patient 
will be marked with the neo^^ gene. In Dr. Rosenberg’s study (37) uptake of neo^ marked 
TIL were demonstrated in malignant melanoma after patients received IV injections of neo^ 
transduced and nontransduced TIL where the proportion of neo*^ transduced cells were as 
low as 1% or 60-200x10® neo^ marked cells. In our studies TIL will be administered by the 
IP and not the IV route. Therefore according to Rosenberg’s results (37) if we inject only 
100x10® neol^ transduced TIL we will be able to detect these cells in vivo. We anticipate that 
at least 10^® CD8+ TIL can be irhected into each patient. Of these lO^® CD8+ cells at least 
half (5x10®) will be from the neo*^ infected bloreactor. With a neo*^ transduction efficiency of 
10% (as we have observed) 5x10® (10% of half 10^®) of the CD8‘‘’ TIL will be marked. Hence, 
5% (5x10® out of 10 1®) of the injected cells will be marked with neo gene. We have 
assumed that a minimum of 10 *® CD8+ TIL can be injected into each patient. Of these 10*® 
CDS'*’ TIL cells, only half (5x10®) will be from the neo*^ infected culture. A minimum neo*^ 
transduction efficiency of 10% has been assured so that a minimum of 5x10® (10% of half of 
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Recombinant DNA Research, Volume 19 
