grow in the absence of IL-2 for greater than 2 weeks, then the N2 marked cells 
would not be administered to the patient. 
C. Analysis of natlent material after infusion 
1) Blood. Mononuclear cells derived from the blood would be 
incubated in TIL medium with G418 in order to detect N2 containing TIL. 
2) Lymph node and tumor biopsy specimens. In patients with 
cutaneous or subdermal tumor or lymph nodes easily accessible, biopsies would be 
performed under local anesthesia at intervals after TIL therapy. These biopsies 
are currently being performed to assess the histologic appearance of tumor in 
treated patients. 
D . Characterization of TIL isolated from •patient material 
1) Direct assay of the cells for vector DNA with Southern blot 
analysis which can detect as few as 5% of positive calls, and polymerize chain 
reaction (PCR) which can detect as few as 1 in 100,000 cells that contain the 
vector DNA. In situ hybridization and anti-NPT antibodies could potentially be 
used to directly examine tissue specimens for N2 vector expression. Methyl- 
cellulose colony assay + G418 could also be utilized to quantitate the number of 
NeoR expressing T cells. 
2) TIL from specimens would be expanded with IL-2, and then analyzed 
as follows: 
a) DNA: Southern blot, PCR 
b) Protein: NPT assay, immunohistochemistry with anti-NPT 
antibody 
c) G418 resistance 
3) Evaluation of the recovered G418- resistant TIL for cytotoxic 
function against tvimor target cells. 
V. PATIENT ELIGIBILITY 
Unchanged from original protocol except that initially the procedure 
described in this Addition would be performed only with selected patients from 
whom tumor biopsies would be easily available. 
VI. TREATMENTS 
Unchanged from original protocol. 
VII. PATIENT EVALUATION 
Unchanged from original protocol except for: 
Recombinant DNA Research, Volume 14 
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