Comparison of Gallium-67 Versus Indium-111. Monoclonal Antibody (96.5, ZME-018) in. Detection of Human Melanoma in Athymic. Mice. S. Ming Chan, Paul B.
Comparison of Gallium-67 Versus Indium-111 Monoclonal Antibody (96.5, ZME-018) in Detection of Human Melanoma in Athymic Mice S. Ming Chan, Paul B. Hoffer, Nada Marie, Sami S. Zoghbi, John M. Kirkwood, Marc S. Ernstoff, Paul H. Duray, and Betsy Gerich Departments of Diagnostic Radiology, Oncology and Pathology, Yale University School of Medicine, New Haven, Connecticut We compared the biodistribution of two radiolabeled, whole, tumor selective monoclonal antibodies ([111ln]96.5, [111ln]ZME-018) to 67Gain nude mice bearing a human melanoma known to express p97 antigen. Localization of gallium was determined 48 hr following i.v. injection. Localization of the radiolabeled antibodies was determined at 3 days and 7 days following i.v. injection. All agents showed more or less similar absolute tumor uptake which varied between 22% and 36% of the injected dose per gram of tumor. Only the tumor uptake of [111ln]96.5 antibody at 7 days was significantly lower than the 67Ga uptake at 48 hr. However, uptake in normal tissues was generally higher for both antibodies at 3 and 7 days than for 67Gauptake at 48 hr. Therefore, the tumor-to-blood ratio for 67Gawas tenfold higher than that for either antibody, the tumor-to-muscle ratio was twofold higher. Bone was the only organ in which the tumor-to-organ ratio was consistently higher with radiolabeled antibody than with 67Ga.The tumor-to-liver and tumor-to-intestine ratios were comparable. Localization of the two tumor selective antibodies was greater than a nonspecific "control antibody" ([111ln]CEA) and change in specific activity from 0.17 mCi/mg to 3.3 mCi/mg did not influence localization. From these animal data it may be anticipated that tumor imaging with [I11ln]96.5 or [l11ln]ZME-018 will not be superior to imaging with 67Gafor detection of melanoma.
J NucÃ-Med 28:1441-1446,1987
its introduction as a tumor imaging agent by Edwards and Hayes in 1969, gallium-67 (67Ga) has been
quine analogs, have also been tested for detection of melanoma but none has proved to be superior to 67Ga.
used to evaluate a variety of malignancies ( I ). Mela noma is one of the tumors in which 67Ga has been used
Recently there has been widespread interest in the use of a variety of radiolabeled monoclonal antibodies to tumor antigens for both detection and therapy of ma lignancy. One of the tumors which has been successfully localized by antibody imaging methods is melanoma (5-9). However, in spite of numerous clinical studies using radiolabeled antibody and at least one study comparing both radiolabeled antibody and 67Ga uptake
successfully for early detection of recurrence (2-4). Nonetheless, [67Ga] citrate is an imperfect agent for tumor imaging. Among its drawbacks in detection of tumor are its nonspecificity, its uptake in normal bone, liver, and spleen which obscures lesions in these organs, its localization in bowel which potentially masks ab dominal lesions, and its occasional failure to localize in lesions which are easily detectable by other modalities. Numerous other agents, such as radiolabeled chloro-
Received Sept. 15, 1986; revision accepted Feb. 4, 1987. For reprints contact: Paul Hoffer, MD, Dept. of Diagnostic Radiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510.
Volume 28 • Number 9 • September 1987
in melanoma in humans (9), no animal studies have been performed to make this comparison. Such studies are critical since even well designed retrospective clini cal studies do not provide reliable comparative data regarding tumor to organ uptake ratios and often in volve differences in imaging techniques and interpretive methods which complicate the interpretation of results. Therefore, we performed the following comparative
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study using mice bearing a human melanoma which is known to accumulate 67Ga and which exhibits high abundance of the melanoma associated p97 antigen (70-72).
radioactivity associated with the tissues was counted in a Beckman gamma 8000 automatic gamma counter. The results were expressed as percent injected dose per gram of tissue (% ID/g). Effect of Specific Activity on the Biodistribution of ('"In] 96.5
MATERIALS
AND METHODS
Radiopharmaceuticals
Indium-111 ('"In) labeled monoclonal antibodies (MoAb) 96.5 (anti p97), ZME-018 (anti-gp240) and anti-CEA were prepared from commercial kits' using the mixed anhydride
The effect of specific activity on biodistribution was deter mined by studying the distribution of ["'In]96.5 in two groups of mice injected with ['"In]96.5 at specific activities of 3.30 fiC't/ng and 0.17 f/Ci/Mg. respectively. The experimental pro cedure was essentially the same as described in the previous section. The mice were killed 72 hr after dose administration.
labeling method. Gallium-67 citrate was used as the radiopharmaceutical+.
Data Analysis
Animals and Tumor
cases where normal distribution may not be assumed, the Wilcoxon nonpaired rank sum test was employed.
Athymic mice (BALB/c-nu/nu) were used as the host ani mals (NIH). The mice are generally 3 to 7 wk of age at the time of tumor implantation (~15 g). Groups of five mice were housed in plastic cages placed in a laminar flow hood, and were allowed pelleted food and water. Human melanoma cells derived from a human tumor specimen were grown in tissue culture. The cells were passed through at least one generation of solid tumor in the nude mouse host prior to study. Solid tumors were produced in the mice by injecting ~5 x IO6 tumor cells in the flank. The tumors were allowed to grow to 4-5 mm in diameter (6-8 wk) prior to experimentation. Immunohistological Studies
Immunohistological studies were performed by using a modification of an avidin-biotin-peroxidase complex (ABC) immunohistochemical method (13-16) to (a) ascertain the presence of p97 and gp240 antigen in the melanoma, and to test for (b) the immunoreactivity of the radiolabeled MoAb, as well as (c) the cross reactivity between an irrelevant (antiCEA) MoAb and the melanoma associated antigen. Cryostat tissue sections were used for the reaction. Sections were in cubated with murine monoclonal 96.5, ZME-018 or anti-CEA as the primary antibody to detect free antigen binding sites still available. Other sections received PBS-Bovine serum al bumin as the primary incubation. The secondary antibody used was horse anti-mouse at a dilution of 1:500. If the ['"In] 96.5, ['"In]ZME-018 or ['"In] anti-CEA had complexed to all binding sites, the secondary antibody would still react to the bound murine monoclonal anti-p97, anti-gp240 or antiCEA. The chromogen substrate was alpha ethyl carbinol which produces a pink to pink-red granular product visible by light microscopy in place of diaminobenzidine because of its rustbrown color which is similar to melanin pigment in melanoma cells. Slides were mounted with an aqueous medium in place of permount due to carbinol sensitivity to alcohol.
Statistical analysis was performed using Student's t-test. In
RESULTS Immunohistological Studies
Nearly 100% of the tumor cells reacted with the nonradiolabeled as well as radiolabeled anti-p97 MoAb, as revealed by the avidin-biotin immunohistochemical technique and as observed in many microscopic fields. However, in one 40x microscopic field, only 50% of the tumor cells exhibited the presence of p97, presum ably indicating tumor heterogeneity in the expression of this antigen. When the irrelevant anti-CEA MoAb was used in the assay,
