A. Preliminary Evaluation and screemng 
Before a patient is entered into the study, the Coinvestigators will review the history, lab data, 
radiographic evaluation, and histopathology . Dr. Holt will confirm that the patient has a malignant 
effusion by examination of the cytology specimens. Selected patients will be admitted to the Clinical 
Research Center where they will have a complete physical exam, blood and urine tests to determine overall 
health. They will also have a baseline chest X-ray, electrocardiogram, and appropriate radiologic 
procedures to assess tumor stage. Patients will be weighed at admission and baseline measurement of 
abdominal girth will be made for patients with malignant ascites. 25 mis of blood will be drawn at 
admission to provide a negative control sample for PCR assays measuring whether retroviral vector is 
present in the peripheral blood following retroviral infusion (blood sample will be split; whole blood will 
be saved for gene assays, and serum will be saved for future immunologic studies). 
B. Evaluations during the Treatment period 
Standard protocol for ascites or pleural effusion: 
Fluid will be removed by paracentesis, thoracentesis, or spinal tap prior to infusion of retroviral 
vector stock. This fluid will be saved as a control for analysis of cytology, DNA, RNA, and 
immunofluorescence studies. Based on the amount of fluid removed in the initial "tap", we will infuse 
approximately 100 mis retroviral vector stock (titers are approximately 7 X 10^ /ml). The usual titer of the 
retroviral stocks for XM6:antifos and XM6:antimyc range from 5-8 x 10^ per millilter, thus a one liter 
infusion into the peritoneal space which deliver approximately 500-800 million viral particles. The amount 
of infusion will be based on the amount of the effusion, clinical judgment, and patient response. The next 
two days (day 2 and day 3) we will withdraw additional ascitic or pleural fluid for the cytologic, 
molecular, and biochemical studies outlined above, and then infuse approximately 100 mis of retroviral 
vector into the site of the effusion. A catheter will be inserted for infusions into selected patients with 
pleural effusions. On day 4 we will obtain a 10 ml fluid sample assess retroviral infection rates and for 
the cytologic, molecular, and biochemical studies described above. A 10 ml blood sample will be taken 
each day for PCR analysis to determine whether the retroviral vector can be detected in the blood. Cell 
samples would be obtained on days 2-4 in order to assess the extent of gene transfer and check for 
apoptosis or differentiation of tumor cells within fluid. If the fluid is substantially absorbed, then we will 
wait to repeat the treatment until the malignant effusion reaccumulates. When and if the malignant fluid 
reaccumulates, then this procedure would be repeated a second and final time. The interval between these 
studies would depend on how rapidly the fluid accumulates. 
Standard protocol for cerebrospinal fluid: 
An omaya reservoir will be placed within the meningeal space and then 3-5 mis of spinal fluid will 
be removed and immediately replaced with retroviral vector stock. The removed fluid will be saved as a 
control for cytology, molecular, and biochemical studies as outlined above. This procedure will be 
repeated each day for 3 days. Fluid samples and a 10 ml blood sample will be taken each day for PCR 
analysis to determine whether the retroviral vector can be detected in the blood. 
Studies to detect spread of retroviral vector: 
We can directly assay gene transfer efficiency in these patients by obtaining fluid from the 
malignant effusion on days following the vector/DNA infusion (or ex vivo infection in the alternate 
protocol) and determining the percent of cells which have integrated the retroviral vector by Southern 
blotting using a globin probe which should provide a ratio of transferred gene: endogenous globin gene 
copy number (See Southern blot in Appendix C). Expression of the target gene will be assayed by in situ 
hybridization and nuclease protection assays to analyze mRNA levels of the antisense mRNA and the 
target mRNA; as well as by immunofluorescent studies analyzing levels of c-fos and c-myc protein in cells 
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