Posted May 26, 2021
Keiran Smalley, Ph.D., H. Lee Moffitt Cancer Center and Research Institute
Metastasis into intracranial structures including the leptomeninges and cerebrospinal fluid (CSF) is one of the most serious complications of advanced melanoma. The leptomeninges comprise the two inner meninges (arachnoid mater and the pia mater) covering the brain and spinal cord. Leptomeningeal metastasis is thought to arise through vascular dissemination of melanoma cells or migration of cells along cranial or spinal nerves. Up to 5%-7% of all melanoma patients will develop leptomeningeal metastasis (LMM), and the prognosis is associated with a mean survival of only 8-10 weeks. In 2017, the Peer Reviewed Cancer Research Program awarded funding for an Idea Award with a Special Focus to Dr. Keiran Smalley at H. Lee Moffitt Cancer Center. Dr. Smalley and his team set out to determine how the brain microenvironment mediates drug resistance in melanoma brain metastasis, examine how the brain shaped the transcriptional profile of the melanoma cells, and identify pathways that astrocytes activate in melanoma cells.
Dr. Smalley’s team collected forty-five CSF samples from 8 patients with and 8 patients without LMM. Samples were collected from the LMM patients throughout the course of treatment following LMM diagnosis; one sample was collected at autopsy. LMM patients underwent combination therapies consisting of checkpoint inhibitors, kinase inhibitors and/or radiation therapies. Most of these 8 LMM patients followed a similar course of BRAF-MEK inhibitor therapy, with one receiving BRAF inhibitor therapy. All but one LMM patient declined despite treatment. One individual responded well to the BRAF-MEK inhibitor therapy and continues to survive more than 35 months after their LMM diagnosis. The control samples were commercially-available CSF from healthy donors as well as samples collected from 8 LMM-negative cancer patients. Samples were analyzed by mass spectrometry and incubated with melanoma cell lines. After incubation, these melanoma cells underwent RNA-Seq analysis as well as functional assays to identify and validate critical pathways.
Dr. Smalley compared the CSF samples from the LMM patients to the CSF from the LMM responder and the non-LMM patients. Results showed 967 proteins differentially expressed in the CSF from the LMM patients who did not respond to therapy and the non-LMM participants, with TGF-β1 identified as a key upstream regulator of these proteins. Differential expression was also noted in the CSF of the LMM responder, with expression changing as the individual responded to therapy. The team confirmed high expression of TGF-β1 in non-responders compared to the responder and controls. In cases where serial samples were available for non-responders, TGF-β1 protein levels increased as the disease progressed. To further explore the role of TGF-β1 in response to therapy, Dr. Smalley treated two melanoma cell lines, WM164 and A375, with BRAF inhibitor in the absence or presence of TGF-β1. He noted that melanoma cells in the presence of both TGF-β1 and BRAF inhibitor showed significantly increased survival compared to cells treated with BRAF inhibitor alone. Mass spectrometry analysis showed that components of the complement pathway, adhesion signaling, and insulin-like growth factor activation pathways were also enriched in CSF samples from the LMM non-responders, which suggests changes in immune regulation, cell proliferation, and migration in LMM patients. In the case of the responder, expression of these pathways decreased as the individual underwent therapy compared to the increase observed during disease progression for the other LMM patients.
Dr. Smalley’s work has demonstrated for the first time that the CSF of patients with LMM is distinct from individuals without LMM. In LMM patients, a number of critical pathways are implicated in immunosuppression, potentially contributing to escape of melanoma cells from therapy. This, combined with the data discerned from the extraordinary responder LMM patient, indicates that the microenvironment of LMM is likely a key regulator of disease progression and therapeutic response, and that CSF markers can potentially be identified for diagnostic and prognostic purposes, providing critical new insights into this rare but devastating complication of melanoma.
Smalley I, Law V, Wyatt C, et al. 2020. Proteomic analysis of CSF from patients with leptomeningeal melanoma metastases identifies signatures associated with disease progression and therapeutic resistance. Clinical Cancer Research. May 01; 26(9): 2163–2175. doi:10.1158/1078-0432.CCR-19-2840.