Up to 14 days later, punch biopsies (3mm) will be obtained of the skin test after 
local infiltration with 1% lidocaine. The punch biopsies will be evaluated to 
establish the nature of the cellular infiltrate. Tissue samples will be processed for 
immunohistology and stained with antisera to detect infiltrating T cells (CD4, 
CD8), B cells (CD20), NK cells (CD56) and macrophages (CD 14, CD 15). These 
will be graded by an independent pathologist. 
9.3.2. Cytotoxic Lymphocytes 
Peripheral blood leukocytes (PBL) will be isolated from heparinized whole blood 
by Ficoll-Hypaque centrifugation. A portion of these cells will be immediately 
tested for cytotoxicity against a battery of targets - K562 (an NK-sensitive cell 
line), M-14 (an NK-insensitive melanoma cell line) and autologous tumor (if a 
cell line or cyropreserved targets are available). - using a standard 4 hr. 
chromium-response assay. Another aliquot of cells will be cultured in the 
presence of 40 u/ml IL-2 for 3-5 days and the same cytotoxicity testing 
performed. 
9.3.3. Tumor Biopsies 
Patients with subcutaneous disease will be asked to consent to have punch 
biopsies of tumor deposits. These biopsies will be performed prior to and at 
monthly intervals after initiation of treatment. The biopsies will be processed 
using the same immunohistochemical stains described in 9.3.1. 
9.3.4. Plasma Cytokine Levels 
The plasma from each blood draw will be stored at - ItfC. IL-2 levels will be 
measured using commercially available ELISA plates (R&D Systems, 
Minneapolis, MN). 
10.0 DOCUMENTATION. RECORD KEEPING. CRFs 
The investigator will maintain adequate records so that the conduct of the study can be 
fully documented and monitored. 
Copies of protocols, case report forms (CRFs), test result originals, all product 
accountability records, correspondence, patient informed consent, and any other 
documents relevant to the conduct of the study will be kept on file by the investigator for 
(five) years after all investigational use of product is discontinued and the FDA is so 
notified or until five years after a Product Licensing Application (PLA/ELA) is 
approved. Study documents will not be destroyed. For FDA inspections, will be 
necessary to have access to complete study patient records, provided that patient 
confidentially is maintained. 
The investigator will obtain a separate release of medical information form to be signed 
by the study patient in order to facilitate access to the patient’s medical records should 
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