Use of fluorescence immunophenotyping combined with in situ hybridization (FICTION) to identify tumor cells in bone marrow of uveal melanoma patients

Abstract

Abstract: : Purpose: To establish a reliable method for the detection of disseminated tumor cells in bone marrow from uveal melanoma patients. Methods: Bone marrow was taken from patients with primary uveal melanoma and healthy persons from the upper iliac crest. Cultured melanoma cells were added in different ratios (5, 10, 20, 40, 60 and 80 %). Cytospins were prepared and immunocytochemistry was performed using antibody MART–1(clone A103). FICTION: Cytospins were first incubated with primary antibody (MART–1). Fluorescence immunophenotyping was performed using the Tyramide amplification technique. Subsequently, interphase in situ hybridisation was performed over night using chromosome 3 centromere specific probe (Vysis). Results: We established MART–1 as a specific marker to identify uveal melanoma cells by immunocytochemistry on cytospins of bone marrow cells from a healthy donor mixed with varying amounts of cultured uveal melanoma cells. However, we occasionally observed positively stained cells in bone marrow of healthy donors. Consequently, to validate cell type we established a technique (FICTION) that allows simultaneous detection of melanoma specific antigens and numerical chromosomal aberrations in cytospins of bone marrow. We determined the chromosome 3 status in interphase nuclei, which is the most frequent chromosomal aberration in metastasizing uveal melanoma. We applied this technique to cytospins of bone marrow cells from a healthy donor mixed with cultured uveal melanoma cells. As a result, we were able to identify tumor cells by a positive MART–1 staining and confirmed the chromosome 3 status by counting the number of chromosome 3 specific signals. Conclusion: Our results show that FICTION facilitates the detection of disseminated tumor cells in bone marrow.

Keywords: melanoma • tumors • oncology