The purpose of this pilot study is to determine the prevalence of markers of chronic and
cycling hypoxia and reactive species stress (oxidative and nitrosative) in the melanoma tumor
microenvironment. The study is based around four cornerstone features of the pathologic
microenvironment - Hypoxia, Reactive Species (reactive oxygen and nitrogen species), HIF-1
and VEGF, which the investigators term the HRHV axis. Patients with in-transit melanoma (AJCC
Stage IIIB or IIIC) (1) will be administered the hypoxia marker drug, EF5, 24 hr prior to
isolated limb infusion (ILI) or hyperthermic isolated limb perfusion (HILP). Tumor biopsies
will be performed just prior to ILI or HILP, at the 30 minute time point during ILI (or 60
minute time point during HILP), AND 24 hours after ILI or HILP. Tissues obtained will be snap
frozen and subsequently analyzed for EF5 binding. Immunohistochemical analysis of a cohort of
immunohistochemical and urine markers that depict the HRHV axis will also be examined. The
association of the markers with the presence of hypoxia, as determined by EF5 positivity,
will be determined. Data from this pilot study will be used to establish the prevalence of
markers of the HRHV axis in melanoma. This information will be crucial for future human
trials in which the HRHV axis is therapeutically targeted.