Flow Cytometric Determination of the Frequency and Heterogeneity of Expression of Human Melanoma-associated Antigens

Abstract

We used flow cytometry to measure the expression of human melanoma antigens on cell suspensions dissociated from metastatic masses. The objective was to study the heterogeneity between tumor samples from different patients and between different tumors excised from a single patient. Fifty-three metastases excised from 34 melanoma patients were analyzed with a panel of nine murine monoclonal antibodies (MOABs). Melanoma cells were stained by an indirect fluorescent method and analyzed on a Coulter EPICS C flow cytometer after gating to exclude tumor-infiltrating leukocytes and dead cells. The most consistently and most strongly expressed antigen was the high-molecular-weight proteoglycan (detected by the MOAB 9.2.27), which was expressed on 95% of the melanoma specimens and by a high proportion of cells within each specimen (mean ± SE, 79.2 ± 5.5). However, strong expression of this antigen was limited to melanoma cells that had been dissociated mechanically and was markedly diminished by exposure to collagenase. Culture of collagenase-dissociated tumor cells for 24 to 48 h resulted in reexpression of the antigen. The expression of other melanoma-associated antigens was not affected by collagenase treatment, but for these antigens there was more variability between cells from an individual tumor and between tumors from different patients. The percentage of enzyme-dissociated tumors considered positive for MOAB binding (defined as at least 10% of cells positive) and the mean ± SE of the percentage of positive cells within a tumor were as follows: MOAB ME-9-61 (antigen, p97) = 84% + (41.2 ± 5.4%); MOAB ME-20.4 (antigen, nerve growth factor receptor) = 40% + (18.7 ± 5.1%); MOAB ME-24 (antigen, ganglioside G_D3) = 84% + (50.8 ± 4.8%); MOAB ME-311 (antigen, ganglioside 9-O-acetyl-G_D3) = 76% + (42.5 ± 5.1%); MOAB ME-361 (antigen, mainly ganglioside G_D2) = 3% + (1.9 ± 0.8%); MOAB 3F8 (antigen, ganglioside G_D2) = 36% (10.5 ± 3.8%); MOAB 14G2a (antigen, ganglioside G_D2) = 86% + (46.0 ± 6.7%); MOAB L243 (antigen, HLA-DR) = 56% + (22.5 ± 5.5%). In 19 cases, we were able to compare the antigenic profiles of two tumors excised from the same patient at different times. Analysis by nonindependent t test showed no significant differences in MOAB binding between the paired tumors. Moreover, linear regression analysis indicated that there was a linear relationship, with a slope approximately = 1, between the percentage of positive cells in Tumor 1 versus Tumor 2. Thus, flow cytometric analysis of cells dissociated from melanoma metastases gives a different perspective than previous studies performed with melanoma cell lines or with tissue sections. In particular, this study suggests that intrapatient heterogeneity of expression of MOAB-defined melanoma antigens may not be as important as previously estimated.