3.0 
SAFETY IMFORMATKW 
3.1 Continued absence of replication competent infectious virus was determined from transfection- 
infection experiments. A neo -containinq vector was transfected into packaging cells; colonies 
were selected with 6418. The supernate were used to infect NIH 3T3 cells. Selection with 6418 
will be done after both 72 hrs and one month to ensure the survival of rare recombinants that do 
not have the neo gene but subsequently infect neo -positive cells. Supernate from the infected NIH 
3T3 cells should not be infectious. These secondary supernate were used to infect naive NIH 3T3 
cells. Lack of infectivity indicates absence of replication competent virus. To date experiments 
have been negative. These studies and the following safety studies will be performed by 
Microbiological Associates, Inc. 
3.2 Sterility will be assured by testing for aerobic and anaerobic bacteria, fungus, and mycoplasma. 
3.3 S7L' assay including 3T3 amplification must be negative. 
3.4 PCR assay for the absence of 4070A envelope gene must be negative. 
3.5 Reverse transcriptase assay must be negative. 
4.0 PATIENT ELIGIBILITY Patient Age: 18 to 80 
4.1 Patients must have histologic proof of non-small cell lung cancer. Patients must be either 
unresectable, unable to receive primary external beam radiation therapy, or have failed primary 
external beam radiation therapy. Patients must have bronchial obstruction as their major problem 
requiring therapy. Patients will be excluded or removed from the study if at any point they 
require systemic chemotherapy for control of progressive metastatic disease. 
4.2 Patients must have an endobronchial tumor accessible by the bronchoscope. There must be some 
clinical evidence of bronchial obstruction. 
4.3 All patients must have a life expectancy of at least 12 weeks and must have a performance status 
of <2 (Zubrod scale. Appendix B). 
4.4 All patients must sign an informed consent indicating that they are aware of the investigational 
nature of this study in keeping with the policies of the hospital. The only acceptable form is 
the one attached at the end of this protocol. 
4.5 A tumor biopsy must show either a K-ras mutation or a p53 mutation by single-strand conformation 
analysis". Material obtained previously and embedded in paraffin may be analyzed. If a new biopsy 
is required, the patient will be entered into the protocol and informed consent obtained if 
protocol entry is the sole reason for the biopsy. K- ras mutations will be determined by specific 
oligonucleotide hybridization to PCR amplified tumor DNA. Mutations of the 2^3 gene will be 
determined by SSCP analysis of exons 5-8 of PCR amplified tumor DNA. 
4.6 Patients will be tested for HIV prior to entry onto the protocol and must be HIV-negative. 
5.0 TREATMENT PLAN 
5.1 Patients will undergo bronchoscopy to assess the degree of obstruction. As much gross tumor as 
possible will be resected endoscopically. Patients may also have had brachytherapy as a tumor 
reduction modality. 
5.2 Patients will undergo bronchoscopy under topical or general anesthesia. A Stifcor^" transbronchi al 
aspiration needle (21g) will be passed through the biopsy channel of the bronchoscope. The 
residual tumor site will be injected with lO' CFU of the appropriate retroviral supernate. The 
volume will be no greater than 10 ml. Protamine will be added at a concentration of Ej/ug/ml . This 
is 0.2X of the amount given Intravenously to reverse heparinization. 
Injections will be circumferential and will be Intratumor and submucosal. The AS-K- ras supernate 
(LNSX-AS-K- ras ) will be used for K- ras mutations and the p53 supernate (LN p53 B) will be used for 
p53 mutations. The injections will be repeated daily for five consecutive days. The treatment 
will be repeated monthly. Treatment will continue as long as there is no tumor progression. After 
one year the patients will be evaluated for continuation of therapy. 
5.3 Patients will wear a surgical mask for 24 hours following injection of the retroviral supernate. 
All medical personnel will wear masks routinely during bronchoscopy and injection of the 
retroviral supernates. Anti -tussi ves will be prescribed as necessary. All patients will be kept 
in isolation during the time they are receiving Injections of the retroviral supernatant and for 
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Recombinant DNA Research, Volume 19 
