I. Patient Selection 
4. Research Design and Methods 
A. Inclusion criteria 
Patients with metastatic breast cancer with spread of tumor into mesothelial-lined spaces: specifically 
cytologically positive malignant ascites, malignant pleural effusion, and carcinomatous meningitis. Female 
patients over age 18 with ability to give informed consent. Patients with ECOG performance status less 
than or equal to 2, life expectancy of greater than 3 months, and who had recovered for at least 4 weeks 
from the morbidity of prior surgery and/or other cancer therapies. 
B. Exclusion criteria 
(1) All medical students are excluded from this study (Vanderbilt policy) 
(2) Pregnant patients 
(3) Patients with acute infectious peritonitis, acute infectious pleuritis, or infectious meningitis 
(4) Patients with congestive heart failure (NYHA claa 4), recent myocardial infarction, 
pulmonary insufficiency, or hematologicsl, renal, and liver dysfunction. 
(5) Patients with less than 3 months expected survival 
(6) Patients requiring anticoagulant or antiplatelet agents 
II. Protocol Design (A summary of the protocol is presented in Table II) 
Patients with metastatic breast cancer who meet the study criteria will be treated with retroviral 
gene therapy by infusion of retroviral vectors into peritoneal, pleural, or meningeal sites containing tumor. 
Retroviral vectors will be manufactured from viral producer cells [39] using serum-free conditions and will 
be tested for sterility, absence of specific pathogens, and an absence of replication-competent retrovirus by 
standard assays (the FDA has specific requirement for biologic therapies of this type and we will comply 
with their standard in our virus production facility in Med Center North). Retrovirus will be stored frozen 
in large aliquots which have been tested according to FDA standards. 
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 chest X-ray, electro- 
cardiogram, and appropriate radiologic procedures to assess tumor stage. 
Patients will spend four days in the Clinical Research Center for the initial 3 infusions of retroviral 
vector. Blood samples will be drawn each day and tested for the presence of retroviral vector by sensitive 
polymerase chain reaction (PCR) -based assays. Patients with malignant pleural effusions will receive a 
flexible intrapleural catheter. An Omaya reservoir will be surgically inserted in those patients with 
carcinomatous meningitis to allow ready access to the CSF. The fluid which is removed will be analyzed 
to determine: 
1. The percentage of cancer cells which are taking up the vector/gene combination: 
by PCR and by in-situ hybridization 
2. The number of cancer cells present in the fluid 
3. Differentiation status of the cells (alcian blue/PAS) 
4. Presence of programmed cell death (ApoTAG and DNA analysis) 
5. Measurement of expression of c-fos or c-myc target gene by slide immunofluorescence or western blot. 
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