III.D.3 
Pathologic evaluation 
Tumor specimens from patients undergoing open resections will be received in Surgical Pathology of the 
University of Pennsylvania. Some of tissue will be fixed in formalin and embedded in paraffin and 
histologic sections will be stained with hematoxylin and eosin. Portions of the tumor will be preserved 
in OTC for immunohistochemical staining, and frozen for PCR analysis of adenovirus DNA (see Table II). 
Immunohistochemical stains for glial fibrillary acidic protein, neurofilament protein and factor VIII, 
adenovirus proteins and HSVf/c protein will be performed. Tumors will be classified according to the 
W.H.O. classification for brain tumors. An estimate of the percentage of tumor composed of proliferating 
vascular tissue will be made. PCR for DNA and RNA will be utilized to detect recombinant virus in the 
surgical pathology brain tumor specimens. A post-mortem examination will be requested on all 
protocol patients. Gross examination and histologic analysis of systemic organs will be performed. 
Analysis of the brain will include an estimate of volume of tumor. Sections of residual tumor and 
normal brain will be snap frozen in liquid nitrogen and stored at -80 °C. Portions of residual tumor 
and surrounding brain will be submitted for histologic processing. In addition to the tumor, sections 
will be taken for histologic analysis from cortex and white matter of the frontal, parietal, temporal and 
occipital lobes, basal ganglia, thalamus, hippocampus, cerebellum, pineal, cervical, thoracic, lumbar 
and sacral levels of the spinal cord and the pituitary gland. In situ hybridization for recombinant virus 
will be performed. 
SPECIMEN IESI FINGINGS/SIGNIFICANCE , 
Tumor tissue 
CSF 
Serum 
Lymphocytes 
H&E 
Immunohistochemistry 
PCR 
Western Blot 
Plaque assay 
Western Blot 
CTL Proliferation 
TABLE II. Pathologic Evaluation. 
% proliferation of vascular tissue 
lymphocytic infiltration histology 
HSVtk protein expression, glial fibrillary 
acidic protein, neurofilament protein, factor 
VIII and adenovirus proteins. 
detection of H5.010RSVf/c 
anti-adenovirus antibody 
detection of viable virus 
anti-adenovirus antibody 
antibodies to HSVf/r 
cytolysis of virus infected cells 
T-cell recognition of adenovirus infected 
cells. , 
III.E Evaluation for Immunological Response 
Serum and cerebrospinal fluid will be collected pre and post therapy for detection of anti-adenovirus 
antibodies by western blot and by neutralizing antibody assay. The patient's humoral immune response 
to adenovirus will be studied by determining titers to adenovirus at various times during the protocol. 
When autologous tumor tissue is available (tumor obtained prior to the gene therapy), cell mediated 
immunity will be studied to see if the patient’s lymphocytes will proliferate in response to tumor cells, 
to adenovirus, or to tumor cells that have been transduced with the virus in vitro. Similarly, cytotoxic 
T-cell responses to the patient's tumor cells will be determined using lymphocytes obtained both pre 
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