Protocol HNS 94-001 
October 4, 1994 
Page 11 
longest and perpendicular dimensions). 
6.6 A pretreatment blood sample will be collected and stored lor analysis of leukocytes and serum. 
7.0 EVALUATION DURING STUDY 
7.1 Patients will have a CBC, platelet count, PT, PTT, SMA-12, electrolytes, and a chest x-ray prior to each 
course of therapy. Serum will be collected pre- and post-treatment for analysis of antibodies to 
adenovirus proteins. 
7.2 History and physical with performance status and weight should be recorded before each course. 
7.3 The tumors will be photographed clinically or endoscopically (depending upon site of disease) at the 
beginning of each course. Tumor measurements are to be recorded before each course for all 
measurable tumors. 
7.4 All relevant information regarding viral dosage, tumor response, laboratory examinations, and 
treatment-related toxicities must be recorded before each treatment is given. 
7.5 Parameters to be Measured In Vitro ? 
7.5.1 Core biopsies or incisional biopsies will be obtained of accessible local and or regional tumor. 
Tumor specimens will be collected 72 hrs. after the last injection of the adenovirus during the 
first treatment cycle. Tissue will be fixed immediately in 4% paraformaldehyde and 0.5% 
glutaraldehyde at 4°C and 1 0% formalin for histopathologic verification and evaluation. This 
will permit extraction of DNA and RNA and permit in situ hybridization. 
7.5.2 Biopsies will be analyzed for incorporation of the transduced gene into the host genomic DNA 
and expression of the transduced gene at the RNA level by standard hybridization 
techniques following polymerase chain reaction. Pre- and post infected tissue biopsies will 
be evaluated histopathologically for any cytomorphologic changes. Tissue blocks will be 
retained for further evaluation of the p53 by immunohistochemistry and in-situ hybridization. 
7.5.3 All patients will be evaluable for response and toxicity following one course of therapy. 
7.6 An autopsy will be requested and immediately performed on all patients enrolled in the protocol who 
die. DNA will be extracted from tumor and normal tissues to determine if the adenovirus genes are 
present and expressed. PCR amplification of specific sequences will be used to determine this. 
7.7 A blood sample will be collected three times at one-half hour intervals following injection of the 
adenovirus. These samples will provide leukocytes to analyze for uptake of adenovirus DNA. Serum 
will be tested for antibodies to adenovirus proteins. This will be done by western blot analysis 
performed by Microbiological Associates, Inc. (Rockville, MD). Patients will be tested monthly during 
treatment, monthly for the first three months following completion of treatment, every three months 
for the remainder of the year following completion of treatment, and then at least yearly thereafter. 
Urine and sputum samples are analyzed daily following virus treatment for adenovirus antibodies. 
7.8 Normal tissue samples will be collected during the follow-up visits and endoscopies. These will 
include samples of non-malignant mucosa, lymphocytes, and germ cells, if possible. These tissues 
will be analyzed for incorporation of the adenovirus. 
7.9 A staging CT scan of the head and neck to evaluate local and regional disease will be obtained on an 
every three month basis during treatment. 
CRITERIA FOR RESPONSE AND TOXICITY 
8 . 1 The graded toxicity scale used in this study has been previously described 29 . Three patients will be 
Recombinant DNA Research, Volume 20 
