Abstract
BACKGROUND: Meningeosis carcinomatosa is a diffuse dissemination of tumor cells in the cerebrospinal fluid (CSF) and/or meninges and occurs in around 5% of patients with malignant melanoma. In previous studies on CSF immune cell subsets an elevated CSF B cell fraction was found in a subset of melanoma patients with leptomeningeal spread. Elevated CSF B cells are mostly associated with neuro-inflammatory diseases and are largely absent during physiological conditions. Hence this study aims to assess if these CSF B cells resemble an intrathecal tumor-specific immune reaction and to test CSF B cell derived recombinant antibodies for tumor specificity from these melanoma patients. METHODS: Three melanoma patients with leptomeningeal spread and a presumable CSF B cell reaction were selected by analyzing CSF parameters including intrathecal immunoglobulin (Ig) synthesis. Single cell sorting of CSF B cells was performed followed by conventional sequencing of Ig heavy and light chain transcripts. 13 recombinant antibodies were generated from CSF B cells and were tested for antigen specificity using antigen microarrays, FACS, ELISA and immunocytochemistry as well as bioinformatic tools for binding predictions. RESULTS: By applying single cell analysis on CSF B cells, representative Ig transcriptome repertoires were obtained. CSF Ig repertoires showed features of a clonal expansion and multiple mutations to germline. These mutations were specifically found in the complementary determining regions suggesting an antigen stimulated affinity maturation within the CSF compartment. By systematic microarrays, the number of potential antigen targets was narrowed down and further evaluated by binding properties of each individual antibody to a well characterized melanoma cell line. Using FACS and immunocytochemistry on the melanoma cell line, antibody binding was confirmed for a subset of antibodies. In custom made ELISA tests, the specific binding of three antibodies to the individual proteins AKR1A1, KIFC3 and DDX53, a known cancer-testis antigen, was detected. Furthermore, potential binding epitopes for these three antibodies were characterized. CONCLUSION: In summary, this study shows evidence for a targeted intrathecal B cell reaction with features of a clonally expanded CSF B cell population in melanoma patients with meningeal carcinomatosis. Specific antibody binding against the targets AKR1A1, KIFC3 and DDX53 was confirmed for three antibodies indicating that these antibodies are indeed tumor-specific and might represent a B cell response against the tumor cells. These antibodies and their corresponding tumor antigens may be extremely useful as they appear to be sufficiently immunogenic to elicit an immune response and thus may be utilized for tumor treatments. GRANTS AND FELLOWSHIPS: This study was funded by the DFG (GZ: KO 4367/5-1; AOBJ: 692835; Projektnummer: 517964704).