Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases

Abstract

Cerebral spinal fluid (CSF) from brain tumor patients contains tumor cellular and cell-free DNA (cfDNA), which provides a less-invasive and routinely accessible method to obtain tumor genomic information. In this report, we used droplet digital PCR to test mutant tumor DNA in CSF of a patient to monitor the treatment response of metastatic melanoma leptomeningeal disease (LMD). The primary melanoma was known to have a BRAF (V600E) mutation, and the patient was treated with whole brain radiotherapy and BRAF inhibitors. We collected 9 CSF samples over 6 months. The mutant cfDNA fraction gradually decreased from 53 % (time of diagnosis) to 0 (time of symptom alleviation) over the first 6 time points. Three months after clinical improvement, the patient returned with severe symptoms and the mutant cfDNA was again detected in CSF at high levels. The mutant DNA fraction corresponded well with the patient's clinical response. We used whole exome sequencing to examine the mutation profiles of the LMD tumor DNA in CSF before therapeutic response and after disease relapse, and discovered a canonical cancer mutation PTEN (R130*) at both time points. The cellular and cfDNA revealed similar mutation profiles, suggesting cfDNA is representative of LMD cells. This study demonstrates the potential of using cellular or cfDNA in CSF to monitor treatment response for LMD.

Keywords: Brain Tumor; Cell-free DNA; Cerebral spinal fluid; Droplet digital PCR; Exome sequencing; Leptomeningeal disease; Melanoma.

Fig. 1

Study design. Samples were collected from a patient undergoing sequential lumbar punctures for the diagnosis and treatment of melanoma leptomeningeal metastases (1). Samples were centrifuged to separate plasma from blood cells and CSF from tumor cells (2). After DNA extraction (3) cellular DNA from CSF and blood underwent exome sequencing to identify tumor mutations. cfDNA from plasma and CSF was utilized to determine the mutant fraction of the known BRAF mutation via ddPCR

Fig. 2

a MRI brain, axial T1 with contrast, showing abnormal contrast enhancement of the right internal auditory canal (red arrow), consistent with cranial nerve involvement by leptomeningeal disease from metastatic melanoma. b CSF cytology (Papanicolaou stain) showing enlarged cell (blue arrow) with enlarged, eccentric nucleoli and intracytoplasmic pigment consistent with metastatic melanoma

Fig. 3

The fraction of mutant BRAF cfDNA in CSF mirrored the clinical symptoms of LMD from metastatic melanoma. a The patient presented to clinic (1) with severe headaches, nausea, and vomiting. Cytology and MRI were consistent with LM metastases of his BRAF-mutated melanoma. The patient started whole brain radiation (RT) and temozolomide (2). He completed RT/temozolomide and began dabrafenib-trametinib, however, the patient’s symptoms persisted. As his symptoms began to improve (3–5), the fraction of mutant DNA in the CSF likewise decreased. When the patient was asymptomatic (6) the BRAF mutation was undetectable in CSF. As the patient was clinically well, additional lumbar punctures were not indicated (6–7). Ninety days later (7), the patient represented to clinic with severe headaches, nausea, and vomiting. He also developed hydrocephalus, requiring placement of a ventriculoperitoneal shunt (9). b Examples of ddPCR plot (time point 8) for CSF cfDNA showing a BRAF mutant fraction of 38 %. Mutants are clustered in the upper left corner with high FAM fluorescent intensities (blue), wild-type is clustered in lower right corner with high HEX fluorescent intensities (green). c Corresponding ddPCR plot (time point 8) for plasma cfDNA did not detect the BRAF mutation, indicating plasma cfDNA was not reflective of the tumor mutation profile within the CNS

Fig. 4

Mutations identified by exome sequencing from pretreatment time point (left) and relapse time point (right). Mutual mutations are shown in blue, nonmutual mutations are shown in grey, and canonical cancer mutations are shown in red