[A case of meningeal melanomatosis diagnosed by immunostaining of cerebrospinal fluid].

A 49-year-old woman was admitted to our hospital with suspected hypertensive encephalopathy. On the basis of MRI showing leptomeningeal enhancement and Class V cytology of the CSF, she was diagnosed as having leptomeningeal carcinomatosis. Although no primary site was detected, a few melanin granules were observed at the third CSF examination. The atypical cells in the CSF demonstrated immunoreactivity for HMB-45 and S-100, which are specific markers of malignant melanoma. There have been few reports of meningeal melanomatosis in Japan. This case illustrates that immunostaining is diagnostically useful in patients with leptomeningeal carcinomatosis from neoplasms with unknown primary sites.

Simultaneous primary cancer occurrence of melanoma and pulmonary adenocarcinoma in leptomeningeal metastases: a case report.

BACKGROUND: Leptomeningeal metastasis (LM) is a predominantly late stage, devastating complication of a variety of malignant solid tumors. Diagnosis relies predominantly on neurological, radiographic, and cerebrospinal fluid (CSF) assessments. Recently, liquid biopsy tests derived from CSF has shown to be a feasible, noninvasive promising approach to tumor molecular profiling for proper brain cancer diagnostic treatment, thereby providing an opportunity for CSF-based personalized medicine. However, LM is typically misleadingly assumed to originate from only one primary tumor type. CASE PRESENTATION: In this case report, we provide first evidence of the co-occurrence of LM originating from more than one primary tumor types. DISCUSSION AND CONCLUSIONS: Based on this patient case profile, the co-occurrence of LM from two or more primary tumor types should be accounted for when deriving diagnostic conclusions from liquid biopsy tests.

Evaluating Circulating Tumor DNA From the Cerebrospinal Fluid of Patients With Melanoma and Leptomeningeal Disease.

Circulating tumor DNA (ctDNA) refers to tumor-derived cell-free DNA that circulates in body fluids. Fluid samples are easier to collect than tumor tissue, and are amenable to serial collection at multiple time points during the course of a patient's illness. Studies have demonstrated the feasibility of performing mutation profiling from blood samples in cancer patients. However, detection of ctDNA in the blood of patients with brain tumors is suboptimal. Cerebrospinal fluid (CSF) can be obtained via lumbar puncture or intraventricular catheter, and may be a suitable fluid to assess ctDNA in patients with brain tumors. We detected melanoma-associated mutations by droplet-digital PCR (ddPCR) and next-generation sequencing in ctDNA obtained from the CSF (CSF-ctDNA) of melanoma patients with leptomeningeal disease. There is a strong correlation between mutation detection by ddPCR, the presence of circulating tumor cells in CSF and abnormalities in the MRI. However, approximately 30% of CSF samples that were negative or indeterminate for the presence of tumor cells by microscopic examination were positive for CSF-ctDNA by ddPCR. Our results demonstrate that CSF is a suitable fluid for evaluating ctDNA and ddPCR is superior to CSF-cytology for analysis of CSF in melanoma patients with leptomeningeal disease.

Two cases of primary leptomeningeal melanomatosis mimicking subacute meningitis.

Primary involvement of leptomeninges with melanocytic tumours is rarely seen and its diagnosis is challenging. Here we summarise two cases of primary leptomeningeal melanomatosis presenting as subacute meningitis. Both cases have pleocytosis and high protein on cerebrospinal fluid analysis, and demonstrated atypical cells on cytology. On magnetic resonance imaging, there is diffuse leptomeningal thickening and avid enhancement of intracranial and intraspinal leptomeninges. One of them demonstrates T1 shortening due to magnetic effects of melanin, the other case is amelanotic and shows hypointensity on precontrast T1-weighted images. Both cases can be diagnosed with biopsy. In conclusion, these cases highlight the importance of the correct interpretation of cytological and magnetic resonance imaging findings in patients with atypical findings.

Quantification of tumor-derived cell free DNA(cfDNA) by digital PCR (DigPCR) in cerebrospinal fluid of patients with BRAFV600 mutated malignancies.

Tumor-derived cell free DNA (cfDNA) can be detected in plasma. We hypothesized that mutated BRAF V600 cfDNA could be quantified in the cerebrospinal fluid (CSF) of patients with central nervous system (CNS) metastases. We collected CSF from patients with BRAF V600E or K-mutated melanoma (N=8) or BRAF V600E mutated Erdheim-Chester Disease (ECD) (N=3) with suspected central nervous system (CNS) involvement on the basis of neurological symptoms (10/11), MRI imaging (8/11), or both. Tumor-derived cfDNA was quantified by digital PCR in the CSF of 6/11 patients (range from 0.15-10.56 copies/µL). Conventional cytology was negative in all patients except in the two patients with markedly elevated levels of tumor-derived cfDNA. In 2 patients with serial measurements, CSF tumor-derived cfDNA levels reflected response to treatment or progressive disease. CSF tumor-derived cfDNA has the potential to serve as a diagnostic tool that complements MRI and may be more sensitive than conventional cytology.

CTLA4 as Immunological Checkpoint in the Development of Multiple Sclerosis.

We investigated a patient who developed multiple sclerosis (MS) during treatment with the CTLA4-blocking antibody ipilimumab for metastatic melanoma. Initially he showed subclinical magnetic resonance imaging (MRI) changes (radiologically isolated syndrome). Two courses of ipilimumab were each followed by a clinical episode of MS, 1 of which was accompanied by a massive increase of MRI activity. Brain biopsy confirmed active, T-cell type MS. Quantitative next generation sequencing of T-cell receptor genes revealed distinct oligoclonal CD4(+) and CD8(+) T-cell repertoires in the primary melanoma and cerebrospinal fluid. Our results pinpoint the coinhibitory molecule CTLA4 as an immunological checkpoint and therapeutic target in MS. Ann Neurol 2016;80:294-300.

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

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.

Extrinsic factors can mediate resistance to BRAF inhibition in central nervous system melanoma metastases.

Here, we retrospectively review imaging of 68 consecutive unselected patients with BRAF V600-mutant metastatic melanoma for organ-specific response and progression on vemurafenib. Complete or partial responses were less often seen in the central nervous system (CNS) (36%) and bone (16%) compared to lung (89%), subcutaneous (83%), spleen (71%), liver (85%) and lymph nodes/soft tissue (83%), P < 0.001. CNS was also the most common site of progression. Based on this, we tested in vitro the efficacy of the BRAF inhibitors PLX4720 and dabrafenib in the presence of cerebrospinal fluid (CSF). Exogenous CSF dramatically reduced cell death in response to both BRAF inhibitors. Effective cell killing was restored by co-administration of a PI-3 kinase inhibitor. We conclude that the efficacy of vemurafenib is variable in different organs with CNS being particularly prone to resistance. Extrinsic factors, such as ERK- and PI3K-activating factors in CSF, may mediate BRAF inhibitor resistance in the CNS.

Cerebrospinal fluid concentrations of vemurafenib in patients treated for brain metastatic BRAF-V600 mutated melanoma.

Anti-BRAF agents, including vemurafenib, have modified the prognosis for patients with melanoma. However, a difference can still be observed between extracerebral and cerebral responses. The aim of this study was to investigate the diffusion of vemurafenib in cerebrospinal fluid (CSF) from patients treated for brain metastatic BRAF-V600 mutated melanoma. Six patients treated with vemurafenib 960 mg twice daily were included. These patients had undergone a lumbar puncture because of suspicions of leptomeningeal metastasis, along with simultaneous blood sampling to measure vemurafenib level. The concentrations of vemurafenib in the CSF and the plasma were measured by high-performance liquid chromatography. The mean plasma and CSF concentrations of vemurafenib were 53.4±26.2 and 0.47±0.37 mg/l, respectively. The mean ratio of the CSF : plasma concentration was 0.98±0.84%. No relationship was found between plasma and CSF concentrations (P=0.8). In conclusion, our preliminary results highlight for the first time a low CSF vemurafenib penetration rate associated with a large interindividual variability in patients treated for metastatic BRAF-V600 mutated melanoma and brain metastases. Further investigations with larger cohorts are required to study the relationship between CSF vemurafenib concentrations and cerebral response.

Leptomeningeal metastases from a primary central nervous system melanoma: a case report and literature review.

Primary central nervous system (CNS) melanoma is a type of rare and aggressive tumor that can easily spread to the leptomeninges, and in fact, leptomeningeal metastasis is one of the most serious complications in patients with this carcinoma. Prognosis is extremely poor if a CNS melanoma has metastasized, and there are no effective treatments. Here, we present a case of a 37-year-old woman who presented with horizontal diplopia and progressive headache. Magnetic resonance imaging findings were consistent with the diagnosis of melanoma. The results of cytological examination of cerebrospinal fluid (CSF) showed malignant cells characteristic of melanoma. No extracranial lesions were observed. All of the available evidence confirmed a diagnosis of leptomeningeal metastases from a primary CNS melanoma. The patient received aggressive treatment, which consisted of concurrent radiotherapy and weekly intra-CSF methotrexate (MTX) followed by adjuvant monthly intra-CSF MTX. Her survival time was 13 months after diagnosis. This case report suggests that the modality of concurrent radiotherapy and weekly intra-CSF MTX followed by adjuvant monthly intra-CSF MTX may be used as the mainstay of treatment for such patients.

[A case of leptomeningeal melanomatosis presenting with right abducens nerve palsy].

A 39 year-old man was admitted to this hospital because of severe headache and vomiting. He had been suffering from lumbago about one month previously, and diplopia ten days previously. The neurological examination revealed disturbance of right eye abduction, no nuchal rigidity. The cerebrospinal fluid (CSF) at the time of admission included erythrocytes (1,490/µl), white blood cell (62/µl) and increased level of protein (531 mg/dl), but no malignant cells were detected. He was treated as meningitis. Cranial magnetic resonance imaging (MRI) demonstrated heterogeneous intensity lesion in the left maxillary sinus and gadolinium enhancement of diffuse meninges and cranial nerves. Spine MRI showed gadolinium enhancement of lumbar spinal meninges and the cauda equina. Biopsy of the lesion in the left maxillary sinus was performed. The pathological findings demonstrated malignant melanoma. Because malignant cells were also observed in CSF, we diagnosed this case as leptomeningeal melanomatosis. Leptomeningeal carcinomatosis should be suspected when headache accompanied with pleomorphic clinical manifestations.

Melanoma brain metastasis globally reconfigures chemokine and cytokine profiles in patient cerebrospinal fluid.

The aggressiveness of melanoma is believed to be correlated with tumor-stroma-associated immune cells. Cytokines and chemokines act to recruit and then modulate the activities of these cells, ultimately affecting disease progression. Because melanoma frequently metastasizes to the brain, we asked whether global differences in immunokine profiles could be detected in the cerebrospinal fluid (CSF) of melanoma patients and reveal aspects of tumor biology that correlate with patient outcomes. We therefore measured the levels of 12 cytokines and 12 chemokines in melanoma patient CSF and the resulting data were analyzed to develop unsupervised hierarchical clustergrams and heat maps. Unexpectedly, the overall profiles of immunokines found in these samples showed a generalized reconfiguration of their expression in melanoma patient CSF, resulting in the segregation of individuals with melanoma brain metastasis from nondisease controls. Chemokine CCL22 and cytokines IL1α, IL4, and IL5 were reduced in most samples, whereas a subset including CXCL10, CCL4, CCL17, and IL8 showed increased expression. Further, analysis of clusters identified within the melanoma patient set comparing patient outcome suggests that suppression of IL1α, IL4, IL5, and CCL22, with concomitant elevation of CXCL10, CCL4, and CCL17, may correlate with more aggressive development of brain metastasis. These results suggest that global immunokine suppression in the host, together with a selective increase in specific chemokines, constitute a predominant immunomodulatory feature of melanoma brain metastasis. These alterations likely drive the course of this disease in the brain and variations in the immune profiles of individual patients may predict outcomes.

Detection and quantification of CSF malignant cells by the CellSearch technology in patients with melanoma leptomeningeal metastasis.

Melanoma is the most frequent solid tumor associated with leptomeningeal metastasis (LM). The usual diagnostic tools, that is, cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium-enhanced MRI of the entire neuraxis both lack effectiveness. The CellSearch Veridex technology for the detection of circulating tumor cells (CTC) in blood was designed for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer, which express EpCAM markers. We have previously adapted this technology to detect malignant cells in the CSF of breast cancer LM. Our objective here was to check if this technology would also allow the detection and the enumeration of CTC in the CSF of melanoma patients presenting with LM although melanoma does not express EpCAM markers. On the occasion of the intrathecal treatment of LM in 2 melanoma patients, 5 mL of CSF and 7.5 mL of blood were collected on CellSave Preservative Tubes and analyzed within 3 days after CSF sampling using a melanoma-dedicated kit. The CellSearch Veridex technology was then adapted to direct enrichment, enumeration, and visualization of melanoma cells in the CSF. CD146+, HMW-MAA+, CD34-, and CD45- cells with typical morphology could be observed and enumerated sequentially with reproducible results, corresponding to CSF melanoma cells (CSFMC). In contrast to the current gold standard cytomorphological analysis, this new approach allowed a precise quantification of CSFMC in all samples concomitantly analyzed. This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of melanoma patients with LM.

Biomarkers of disease: cerebrospinal fluid vascular endothelial growth factor (VEGF) and stromal cell derived factor (SDF)-1 levels in patients with neoplastic meningitis (NM) due to breast cancer, lung cancer and melanoma.

BACKGROUND: Breast cancer, lung cancer and melanoma metastasize to the meninges in 5-15% of patients. The identification of specific biomarkers of disease may allow for earlier diagnosis and treatment. Preclinical evidence suggests the possible relevance of SDF-1 and VEGF in the homing and neoangiogenesis of metastases. We chose to measure these molecules in the cerebrospinal fluid (CSF) of melanoma, breast, and lung cancer patients being evaluated for neoplastic meningitis (NM). MATERIALS AND METHODS: We collected CSF from patients with these cancers who were being evaluated for possible NM. CSF was assayed for SDF-1 and VEGF levels using Enzyme-linked Immunosorbent Assay (ELISA) assays. RESULTS: CSF samples from 89 patients met criteria for analysis, including 41 with breast cancer, 35 with lung cancer and 13 with melanoma. Twenty-five percent (22/89) of all samples were positive for malignant cells; 8/41 (20%) from breast cancer, 10/35 (29%) from lung cancer and 4/13 (31%) from melanoma. CSF VEGF levels were available from 83 patients, and were elevated (>20 pg/ml) in 15/22 (68%) of patients with positive CSF cytology and normal (<20 pg/ml) in 59/61 (97%) of patients with negative CSF cytology. The two patients with negative CSF cytology who also had elevated CSF VEGF levels had MRI evidence of NM. CSF SDF-1 levels were available from 81 patients, and were elevated (>950 pg/ml) in 11/18 (61%) of patients with positive CSF cytology and normal (<950 pg/ml) in 57/63 (90%) of patients with negative CSF cytology. CONCLUSIONS: Elevated CSF levels of VEGF are sensitive and highly specific for the diagnosis of NM from breast cancer, lung cancer and melanoma, and may serve as a useful biomarker of NM in high risk patients. CSF SDF-1 levels add little to the diagnostic information provided by CSF VEGF. Evaluation of CSF VEGF levels as a trigger for early treatment in high risk breast cancer, lung cancer and melanoma patients at risk for NM, is warranted.

Neurocutaneous melanosis associated with malignant leptomeningeal melanoma in an adult: clinical significance of 5-S-cysteinyldopa in the cerebrospinal fluid---case report.

A 35-year-old male presented with a variant of neurocutaneous melanosis with leptomeningeal malignant melanoma. He had three pigmented nevi from birth. He suffered diplopia followed by headache. T1-weighted magnetic resonance (MR) imaging revealed hydrocephalus and a small linear hyperintense lesion in the right frontal cortex. Several parts of the cortical sulci and the brain surface were slightly enhanced by gadolinium. Ventriculoperitoneal shunting was performed and extensive pigmented leptomeninges were recognized. Open biopsy established the diagnosis of leptomeningeal malignant melanoma. Combined chemoimmunotherapy was repeated every other month with monitoring of the 5-S-cysteinyldopa (5-S-CD) level in the cerebrospinal fluid (CSF). The 5-S-CD level decreased after each treatment, but the basal level steadily increased prior to the next treatment. Two years after the onset, he showed paraplegia caused by an extramedullary mass at the T-6 level. MR imaging showed that melanoma had involved the entire subarachnoid space including the whole spine. He underwent emergent removal of the spinal tumor and showed transient marked improvement. Further intensive chemotherapy was given. However, he died 31 months after the onset of massive proliferation of intracranial leptomeningeal melanoma. Measurement of CSF 5-S-CD levels is valuable for evaluating the therapeutic efficacy and for monitoring the progression of melanoma.