1.2. a. BIOPSY OF THE VACCINE SITE. Preclinical data show this to 
be one of the most sensitive measurements of the effect of local tumor 
production of GM-CSF. Histologically, the response to subcutaneously injected 
irradiated non-transduced tumor varies, depending on the " immunogenicity " of 
the tumor. The histopathologic response is unmistakably different when the 
tumor is transduced to produce GM-CSF, with the former showing a surrounding 
cuff of lymphocytes, while the latter has a profound infiltration of 
monocytes, macrophages, eosinophils, and later, lymphocytes and PMNs. The 
kinetics of this response in the mouse are that by 5 to 7 days there is little 
viable tumor left. It is acknowledged that at Dose Levels 1 and 2, where the 
dose is divided into four injections per cycle, the biopsy of the vaccine site 
(once on day three and again on day seven) is in a sense "removing" a modest 
fraction of the therapeutic agent. This is felt to be justifiable on several 
accounts. Firstly, the punch biopsies are far from excisional, leaving behind 
a sizeable component of the vaccination at that site. Secondly, if the 
kinetics of the human response mirror that seen in the mouse, by the time of 
the day seven biopsy, the critical events of the antigen processing and 
presentation may have already occurred. Finally, in all Dose Levels, the 
majority of the vaccine is left undisturbed at the other sites for each cycle, 
and the amount removed is relatively constant between patient groups. 
7.2. b. DTH RESPONSE. The delayed type hypersensitivity response 
is a well characterized in vivo measurement of antigen specific T cell 
mediated responses. McCune and colleagues reported a correlation between the 
development of a DTH response to autologous tumor and clinical response in 
patients with advanced renal cell carcinoma. 37 In addition to the standard 
measurements of induration and erythema, we propose to biopsy the DTH site. 
Our preclinical data shows that the histologic appearance of the response to 
the "priming" GM-CSF producing vaccine is dramatically different than that of 
the contralateral non-GM-CSF producing "challenge" site. This qualitative 
difference between the histology of the vaccine site and the challenge site 
was also noted in other tumor/cytokine combinations. Some vaccines (such as 
B16 melanoma/IL-2 ) showed impressive infiltration of the vaccine site, but 
very little infiltrate at the site of the tumor challenge. The response to 
the non-cytokine producing tumor challenge apparently more closely reflects 
the nature of the systemic immune response which has the greatest relevance to 
activity at sites of distant metastases. Ideally, it would be helpful to have 
biopsies of actual metastases, but in this patient population this could incur 
the risk of serious morbidity. The biopsy of the DTH response (i.e. 
irradiated non-transduced autologous tumor injected subcutaneously) may 
provide similar information with substantially less risk to the patient. 
Given the possibility that non-transduced irradiated autologous tumor 
may be significantly immunogenic, it must be acknowledged that the DTH testing 
may alter that which it is intended to measure. In fact, for patients 
randomized to no GM-CSF transduction at Dose Level one, the "pretreatment DTH" 
testing amounts to 25% of the dose of the first cycle. While this dose may 
have some priming effect, it is the change in the DTH response that occurs 
with subsequent vaccination that will be the critical determinant of 
immunological activity. Also, our preclinical data suggest that total cell 
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Recombinant DNA Research, Volume 17 
