Implementation of cell‑free tumor DNA sequencing from the cerebrospinal fluid to guide treatment in a patient with primary leptomeningeal melanoma: A case report

  • Authors:
    • Johannes C. Melms
    • Ka‑Wai Ho
    • Rohit Thummalapalli
    • Janice Tyler
    • Titus Josef Brinker
    • Veena Singh
    • Soma Sengupta
    • James Mier
    • Benjamin Izar
  • View Affiliations

  • Published online on: May 9, 2018
  • Pages: 58-61
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Primary leptomeningeal melanoma (PLM) is a rare type of cancer that represents a major clinical and molecular diagnostic challenge. A definitive diagnosis requires consistent magnetic resonance imaging findings and cerebrospinal fluid (CSF) cytology. Due to the small number of malignant cells in the CSF, routine testing for mutations in the BRAF gene is difficult, which prevents the stratification of these patients to potentially beneficial therapies. We herein present the case of a 62‑year old man with CSF cytology indicating PLM, where BRAF mutation testing, from cell‑free (cf) tumor DNA isolated from the CSF and plasma was implemented to guide clinical decision making. Testing for BRAFV600E mutation from the CSF and plasma was technically feasible, yielded concordant results, and guided the treatment for this patient. This case suggests that mutation testing of cfDNA isolated from the CSF is technically feasible and may guide therapy in cases where a tissue diagnosis is not possible for PLM and other malignancies with defined oncogenic driver mutations.


Melanoma is the deadliest form of skin cancer and continues to have an increasing incidence in the last decades (1). About ~50% of melanomas harbor a T to A substitution in codon 600 of the BRAF gene, resulting in a substitution of Valine to Glutamic Acid (BRAFV600E), causing tonic activation of the RAF/MEK/ERK pathway, proliferation, and cell survival (2,3). While a combination of selective RAF/MEK inhibitors results in brisk responses in most patients with BRAFV600E, treatment with these drugs in BRAF wild-type (WT) tumors leads to paradoxical activation of the pathway with the potential to accelerate tumor growth (4,5). Determining the BRAF mutation status is therefore critical prior to initiation of RAF/MEK-inhibitors. In contrast, immune checkpoint inhibitors, which are monoclonal antibodies that target CTLA-4 (i.e., ipilimumab) or the PD-1/PD-L1 axis (e.g., nivolumab or pembrolizumab) exhibit activity irrespective of the BRAF mutation status, and are therefore the preferred first-line therapy for patients with BRAF WT melanoma (68). Testing for the BRAF mutation is usually performed on a tissue biopsy, such as a core needle biopsy. However, there are instances, in which potentially significant morbidity prohibits procedures for obtaining tissue. Primary leptomeningeal melanoma (PLM) is a rare and very aggressive type of melanoma with an estimated frequency of 1 in 10 million individuals (912). Due to its localization involving the letptomeninges, biopsies cannot be easily performed, which may limit potential therapeutic benefits for these patients. Here, we describe a case of a patient with PLM who underwent BRAF mutation testing from cell-free DNA (cfDNA) isolated from cerebrospinal fluid (CSF) to guide therapy choices.

Case report

A 62-year-old Caucasian man with a past medical history of essential hypertension and obstructive sleep apnea was admitted to Beth Israel Deaconess Medical Center with altered mental status, headaches and gait difficulties in March of 2016. Three months prior to presentation, the patient had noticed lower back pain with radiation to the buttocks. Magnetic resonance imaging (MRI) without contrast of the lumbar spine at that time was unrevealing. Over the following 6 weeks, the patient developed worsening gait difficulties and intermittent confusion and, 3 days prior to presentation, he developed headaches and was persistently confused.

On arrival to our emergency room, the patient was somnolent and only oriented to name. The vital signs were notable for a temperature of 102°F, heart rate 74 beats/min, blood pressure 162/98 mmHg, respiratory rate 20 breaths/min, and oxygen saturation 97% at ambient air. Physical examination revealed somnolence with responses only to noxious stimuli, nuchal rigidity with a positive Brudzinski sign, and bilateral papilledema. An immediate non-contrast head computed tomography (CT) scan showed extensive communicating hydrocephalus and transependymal flow. The patient underwent a large-volume lumbar puncture where the opening pressure was 34 cm H2O, with subsequent mental status improvement. A complete cell count and chemistry of the CSF is summarized in Table I. The patient was admitted to the neurological intensive care unit for further care.

Table I.

CSF cell count and chemistry from two LPs performed on hospital days 1 and 4.

Table I.

CSF cell count and chemistry from two LPs performed on hospital days 1 and 4.

LP no.WBC (/µl)RBC (/µl)PMN (%)LYM (%)MONO (%)MACRO (%)OTHER (%)Glucose (mg/dl)Protein (mg/dl)
1 (HD1)2816,750424871460516
2 (HD4)57,950757623653459
Reference range<500000045–8015–45

[i] CSF, cerebrospinal fluid; LP, lumbar puncture; HD, hospital day; WBC, white blood cells; RBC, red blood cells; PMN, polymorphonuclear leukocytes; LYM, lymphocytes; MONO, monocytes; MACRO, macrophages.

An MRI scan of the head and spine with and without contrast revealed diffuse leptomeningeal enhancement involving intracranial and spinal meninges on post-contrast T1-weighted imaging, as well as communicating hydrocephalus. There was no parenchymal central nervous system (CNS) involvement (Fig. 1A-C). A CT scan of the chest, abdomen and pelvis did not reveal evidence of visceral metastatic disease. A full skin examination by a dermatologist reported no evidence of a primary cutaneous melanoma, and the ophthalmic examination did not reveal any suspicious lesions. Cytology of the CSF revealed malignant cells with strong staining for human melanoma black-45 (HMB-45), confirming a diagnosis of malignant melanoma (Fig. 1D and E).

Overall, this presentation was consistent with a primary leptomeningeal melanoma (PLM). The patient was treated with dexamethasone and required emergent placement of an external ventricular drain (EVD) due to interval worsening mental status in the setting of hydrocephalus. CSF was collected for isolation of cfDNA and sequencing of the BRAF gene. Plasma was collected at the same time for cfDNA sequencing. BRAF mutation testing of CSF and plasma was performed using the Biocept Target Selector assay.

The patient received five fractions of whole-brain radiation therapy (2,000 cGy) and palliative radiation of the spine from level T12 to S3. He had significant improvement of his neurological symptoms, allowing for removal of the EVD on day 12 of his hospitalization, and was discharged from the hospital on day 20.

Although previous efforts have used targeted next-generation sequencing to evaluate small panels of genes involved in melanoma biology, including BRAF and NRAS (13), only mutant BRAF is a currently actionable target and may help guide the choice of targeted therapy vs. immunotherapy. Sequencing of cfDNA revealed wild-type (WT) BRAF gene in both the CSF and plasma. Based on this finding, treatment with either ipilimumab, a PD-1 checkpoint inhibitor, or temozolomide was discussed. Given the patient's good clinical status, treatment with ipilimumab was initiated, with a plan to administer four cycles, potentially followed by PD-1 checkpoint blockade. Although the patient received his first infusion without treatment-related complications, the course was complicated by the development of a pulmonary embolism, which delayed a planned second infusion. Five weeks after the first ipilimumab infusion (~9 weeks after the initial diagnosis), the patient developed rapidly progressing confusion and gait instability with worsening hydrocephalus and succumbed to the disease 3 days later.


Primary leptomeningeal melanoma (PLM) is a very rare type of cancer that is considered to arise from melanocytes in the pia and arachnoid (10). Diagnostic criteria for PLM have been suggested (9), including hyperintensity of the meninges on T1-weighted MRI and cytology with positive immunostaining for lineage-specific HMB-45 and S-100 (1416).

In ~25% of patients, PLM is associated with giant melanocytic nevi, which frequently carry treatment-sensitizing oncogenic driver mutations (17,18) including BRAFV600E and NRASQ61. Among patients with metastatic cutaneous melanoma, ~50% harbor sensitizing BRAF mutations, most commonly BRAFV600E. Treatment with RAF or RAF/MEK-inhibitors in this subset of patients has resulted in unprecedented response rates and improvement of progression-free and overall survival (4,19). However, patients with wild-type BRAF melanoma are not candidates for RAF/MEK inhibition, as BRAF inhibitors may promote growth of BRAF-WT cells and further exacerbate the disease (20), highlighting the importance of targeted BRAF testing in this patient. In patients with BRAF-WT melanoma, first-line immunotherapies are currently under investigation as an alternative strategy. To this end, the Food and Drug Administration has approved immunotherapies, such as the CTLA-4 inhibitor ipilimumab and PD-1 checkpoint inhibitors, including nivolumab and pembrolizumab, as first-line therapies for patients with metastatic melanoma. Single-agent treatment with any of these compounds or combinations of ipilimumab and PD-1 inhibitors produce deep and long-lasting responses in a subset of patients (68,21), including those with leptomeningeal disease (22).

The ideal choice of first-line therapy, RAF/MEK-inhibitors or immunotherapies, in patients with BRAF-mutant melanoma remains unclear and is mostly guided by the clinical setting. For example, in patients with rapidly progressing BRAF-mutant melanoma, treatment with BRAF/MEK inhibitors may induce faster responses and is the preferred treatment modality. In patients without sensitizing BRAF mutations (BRAF-WT), as in the present case, immunotherapy is the first-line treatment.

Understanding the molecular profile of these tumors is crucial for employing treatments such as targeted therapies or immune checkpoint inhibitors, which may induce dramatic responses in leptomeningeal melanoma (22,23). However, testing from malignant CSF with as few as 1 malignant cell per µl, as in the case presented here, is challenging with current approaches. We herein report the successful use of targeted BRAF sequencing of cfDNA isolated from the CSF as well as the plasma in a patient with PLM. The results from BRAF mutation testing were instrumental in selecting the treatment for this patient, given the potential harm in treating a BRAF-WT patient with RAF/MEK inhibitors. Recent reports indicating the feasibility of molecular profiling from CSF (2427) have mostly focused on primary CNS tumors. With regard to cfDNA sequencing from CSF for melanoma, previous reports have focused only on monitoring treatment response in metastatic lesions for which the genomic status of the primary melanoma lesion was known (28,29). In contrast, our case displays the utility of using cfDNA to guide treatment choice in a primary leptomeningeal melanoma for which no genetic information was available. This study indicates that rapid targeted sequencing of cfDNA from the CSF is clinically feasible and should be considered for guiding treatment in patients in whom a tissue biopsy cannot be obtained, including those with PLM and leptomeningeal metastatic disease.


We thank Lyle Arnold, PhD and Cecile Rose Vibat, PhD from Biocept Inc. for providing technical support.


BI is supported by the National Cancer Institute (K08CA222663) and the Ludwig Center for Cancer Research at Harvard.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

JCM, KH, TJB, SS, JM and BI took clinical care of the patient. JT provided pathology slides. JCM, RT and BI wrote the paper. All authors read, reviewed and approved the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

The family agreed to publication of the case and material presented here.

Competing interests

VS is an employee of BioCept Inc. The other authors declare that they have no competing interests.



Siegel RL, Miller KD and Jemal A: Cancer statistics, 2017. CA Cancer J Clin. 67:7–30. 2017. View Article : Google Scholar : PubMed/NCBI


Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, Nickerson E, Auclair D, Li L, Place C, et al: A landscape of driver mutations in melanoma. Cell. 150:251–263. 2012. View Article : Google Scholar : PubMed/NCBI


Cancer Genome Atlas Network: Genomic classification of cutaneous melanoma. Cell. 161:1681–1696. 2015. View Article : Google Scholar : PubMed/NCBI


Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF, Nolop K and Chapman PB: Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 363:809–819. 2010. View Article : Google Scholar : PubMed/NCBI


Poulikakos PI, Zhang C, Bollag G, Shokat KM and Rosen N: RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 464:427–430. 2010. View Article : Google Scholar : PubMed/NCBI


Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, et al: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 363:711–723. 2010. View Article : Google Scholar : PubMed/NCBI


Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P, et al: Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 373:23–34. 2015. View Article : Google Scholar : PubMed/NCBI


Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, Daud A, Carlino MS, McNeil C, Lotem M, et al: Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 372:2521–2532. 2015. View Article : Google Scholar : PubMed/NCBI


Hayward RD: Malignant melanoma and the central nervous system. A guide for classification based on the clinical findings. J Neurol Neurosurg Psychiatry. 39:526–530. 1976. View Article : Google Scholar : PubMed/NCBI


Rosenthal G, Gomori JM, Tobias S, Diment J and Shoshan Y: Unusual cases involving the CNS and nasal sinuses: Case 1. Primary leptomeningeal melanoma. J Clin Oncol. 21:3875–3877. 2003. View Article : Google Scholar : PubMed/NCBI


Paulus W and Hasselblatt M: TumorenNeuropathologie. Springer; Berlin-Heidelberg: pp. 481–549. 2012


Hsieh YY, Yang ST, Li WH, Hu CJ and Wang LS: Primary leptomeningeal melanoma mimicking meningitis: A case report and literature review. J Clin Oncol. 33:e57–e61. 2015. View Article : Google Scholar : PubMed/NCBI


van de Nes J, Gessi M, Sucker A, Möller I, Stiller M, Horn S, Scholz SL, Pischler C, Stadtler N, Schilling B, et al: Targeted next generation sequencing reveals unique mutation profile of primary melanocytic tumors of the central nervous system. J Neurooncol. 127:435–444. 2016. View Article : Google Scholar : PubMed/NCBI


Wick MR, Swanson PE and Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol. 15:201–207. 1988. View Article : Google Scholar : PubMed/NCBI


Tosaka M, Tamura M, Oriuchi N, Horikoshi M, Joshita T, Sugawara K, Kobayashi S, Kohga H, Yoshida T and Sasaki T: Cerebrospinal fluid immunocytochemical analysis and neuroimaging in the diagnosis of primary leptomeningeal melanoma. Case report. J Neurosurg. 94:528–532. 2001. View Article : Google Scholar : PubMed/NCBI


Sagiuchi T, Ishii K, Utsuki S, Asano Y, Tsukahara S, Kan S, Fujii K and Hayakawa K: Increased uptake of technetium-99m-hexamethylpropyleneamine oxime related to primary leptomeningeal melanoma. AJNR Am J Neuroradiol. 23:1404–1406. 2002.PubMed/NCBI


Hoffman HJ and Freeman A: Primary malignant leptomeningeal melanoma in association with giant hairy nevi. J Neurosurg. 26:62–71. 1967. View Article : Google Scholar : PubMed/NCBI


Salgado CM, Basu D, Nikiforova M, Bauer BS, Johnson D, Rundell V, Grunwaldt LJ and Reyes-Múgica M: BRAF mutations are also associated with neurocutaneous melanocytosis and large/giant congenital melanocytic nevi. Pediatr Dev Pathol. 18:1–9. 2015. View Article : Google Scholar : PubMed/NCBI


Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, Demidov LV, Hassel JC, Rutkowski P, Mohr P, et al: Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 367:107–114. 2012. View Article : Google Scholar : PubMed/NCBI


Medina TM and Lewis KD: The evolution of combined molecular targeted therapies to advance the therapeutic efficacy in melanoma: A highlight of vemurafenib and cobimetinib. Onco Targets Ther. 9:3739–3752. 2016.PubMed/NCBI


Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, et al: Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med. 369:134–144. 2013. View Article : Google Scholar : PubMed/NCBI


Margolin K, Ernstoff MS, Hamid O, Lawrence D, McDermott D, Puzanov I, Wolchok JD, Clark JI, Sznol M, Logan TF, et al: Ipilimumab in patients with melanoma and brain metastases: An open-label, phase 2 trial. Lancet Oncol. 13:459–465. 2012. View Article : Google Scholar : PubMed/NCBI


Wilgenhof S and Neyns B: complete cytologic remission of V600E BRAF-mutant melanoma-associated leptomeningeal carcinomatosis upon treatment with dabrafenib. J Clin Oncol. 33:e109–e111. 2015. View Article : Google Scholar : PubMed/NCBI


Pan W, Gu W, Nagpal S, Gephart MH and Quake SR: Brain tumor mutations detected in cerebral spinal fluid. Clin Chem. 61:514–522. 2015. View Article : Google Scholar : PubMed/NCBI


Wang Y, Springer S, Zhang M, McMahon KW, Kinde I, Dobbyn L, Ptak J, Brem H, Chaichana K, Gallia GL, et al: Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci USA. 112:9704–9709. 2015. View Article : Google Scholar : PubMed/NCBI


De Mattos-Arruda L, Mayor R, Ng CK, Weigelt B, Martínez-Ricarte F, Torrejon D, Oliveira M, Arias A, Raventos C, Tang J, et al: Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat Commun. 6:88392015. View Article : Google Scholar : PubMed/NCBI


Pentsova EI, Shah RH, Tang J, Boire A, You D, Briggs S, Omuro A, Lin X, Fleisher M, Grommes C, et al: Evaluating cancer of the central nervous system through next-generation sequencing of cerebrospinal fluid. J Clin Oncol. 34:2404–2415. 2016. View Article : Google Scholar : PubMed/NCBI


Li Y, Pan W, Connolly ID, Reddy S, Nagpal S, Quake S and Gephart MH: Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases. J Neurooncol. 128:93–100. 2016. View Article : Google Scholar : PubMed/NCBI


Momtaz P, Pentsova E, Abdel-Wahab O, Diamond E, Hyman D, Merghoub T, You D, Gasmi B, Viale A and Chapman PB: Quantification of tumor-derived cell free DNA(cfDNA) by digital PCR (DigPCR) in cerebrospinal fluid of patients with BRAFV600 mutated malignancies. Oncotarget. 7:85430–85436. 2016. View Article : Google Scholar : PubMed/NCBI

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Melms, J.C., Ho, K., Thummalapalli, R., Tyler, J., Brinker, T.J., Singh, V. ... Izar, B. (2018). Implementation of cell‑free tumor DNA sequencing from the cerebrospinal fluid to guide treatment in a patient with primary leptomeningeal melanoma: A case report. Molecular and Clinical Oncology, 9, 58-61.
Melms, J. C., Ho, K., Thummalapalli, R., Tyler, J., Brinker, T. J., Singh, V., Sengupta, S., Mier, J., Izar, B."Implementation of cell‑free tumor DNA sequencing from the cerebrospinal fluid to guide treatment in a patient with primary leptomeningeal melanoma: A case report". Molecular and Clinical Oncology 9.1 (2018): 58-61.
Melms, J. C., Ho, K., Thummalapalli, R., Tyler, J., Brinker, T. J., Singh, V., Sengupta, S., Mier, J., Izar, B."Implementation of cell‑free tumor DNA sequencing from the cerebrospinal fluid to guide treatment in a patient with primary leptomeningeal melanoma: A case report". Molecular and Clinical Oncology 9, no. 1 (2018): 58-61.