within malignant fluids. Note that these analyses will not be limiting to the intended target cell populations, 
since we can also determine whether the antisense mRNA is expressed within normal cells within the fluid: 
a direct test to determine whether the transferred gene is selectively targeted for the tumor cells. The 
Southern blot should be able to detect the percent of cells which have been infected by the virus as long as 
at least 5% of the cells have been infected. Similarly, nuclease protection assays and immunofluroescent 
assays have been capable of detecting expression in 3-5% of cells. If greater sensivity is needed, then we 
will employ the PCR- based tests which we have designed to detect the presence of retroviral vector DNA 
in peripheral blood. We estimate the sensivity of the PCR assay to be able to detect one integration in 10^ 
cells. 
C. Evaluation following the Treatment Period 
1. Initial followup: 
Patients will be seen as outpatients at 2 week intervals during the first two months and on a 
monthly basis during the remainder of the first year. Blood samples to analyze for the presence of 
retroviral vector in the peripheral blood will be performed at the 1 week, 2 week, 4 week, and 8 week 
visits. If any of these tests are positive then we will continue screening at later visits. If fluid reaccumulates 
and patients require drainage then fluid will be obtained and cytologic, molecular, and biochemical studies 
repeated. 
2. Yearly followup: 
Patients will be asked to return at least once a year for the rest of their life for retroviral gene 
therapy safety monitoring. Blood samples will be drawn at this time for PCR studies to analyze whether 
the retroviral vector is present in peripheral blood cells. Fluid will be tapped when clinically indicated. 
3. Autopsy: 
We will try to obtain consent for a complete autopsy on any patient who dies during or after 
the study. Tissues from all major organs: including the ovaries, bone marrow, liver; and from the lining 
of the involved mesothelial-lined cavity will be obtained for cytologic, molecular, and biochemical 
analyses. PCR, in situ hybridization, and Southern blot studies will be performed to determine whether 
remaining cells are present which have integrated and/or express the retroviral viral vector. We will 
particularly search for integration and expression into non-tumorous cells. 
4. It is understood thast the performance of this study is subject to factors including but not limited to: 
patient compliance, scheduling difficulties, clinical judgement of the investigators, or clinical judgement of 
the patient care physician. In this case, a test may not be done in an individual instance with no violation of 
the protocol. However any systemic or significant modification of the original protocol, whether related to 
patient safety or not, will be submitted to the Vanderbilt IRB for approval. 
D. Criteria for response 
1. Non-responders: 
Patients with less than a 25% decrease in abdominal girth above control for body surface area or 
25% decrease in effusion volume (judged by X-ray) and with no reduction of malignant cells within the 
fluid. 
2. Minimal responders: 
Patients with a 25-49% decrease in abdominal girth or similar decrease in effusion volume; and 
with a greater than 50% reduction of the number of malignant cells within the fluid. 
Recombinant DNA Research, Volume 20 
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