Liquid biopsy of cerebrospinal fluid for MYD88 L265P mutation is useful for diagnosis of central nervous system lymphoma.

The current standard of diagnosing central nervous system (CNS) lymphoma is stereotactic biopsy, however the procedure has a risk of surgical complication. Liquid biopsy of the CSF is a less invasive, non-surgical method that can be used for diagnosing CNS lymphoma. In this study, we established a clinically applicable protocol for determining mutations in MYD88 in the CSF of patients with CNS lymphoma. CSF was collected prior to the start of chemotherapy from 42 patients with CNS lymphoma and matched tumor specimens. Mutations in MYD88 in 33 tumor samples were identified using pyrosequencing. Using 10 ng each of cellular DNA and cell-free DNA (cfDNA) extracted from the CSF, the MYD88 L265P mutation was detected using digital PCR. The conditions to judge mutation were rigorously determined. The median Target/Total value of cases with MYD88 mutations in the tumors was 5.1% in cellular DNA and 22.0% in cfDNA. The criteria to judge mutation were then determined, with a Target/Total value of 0.25% as the cutoff. When MYD88 mutations were determined based on these criteria, the sensitivity and specificity were 92.2% and 100%, respectively, with cellular DNA; and the sensitivity and specificity were 100% with cfDNA. Therefore, the DNA yield, mutated allele fraction, and accuracy were significantly higher in cfDNA compared with that in cellular DNA. Taken together, this study highlights the importance of detecting the MYD88 L265P mutation in cfDNA of the CSF for diagnosing CNS lymphoma using digital PCR, a highly accurate and clinically applicable method.

Circulating tumour DNA from the cerebrospinal fluid allows the characterisation and monitoring of medulloblastoma.

The molecular characterisation of medulloblastoma, the most common paediatric brain tumour, is crucial for the correct management and treatment of this heterogenous disease. However, insufficient tissue sample, the presence of tumour heterogeneity, or disseminated disease can challenge its diagnosis and monitoring. Here, we report that the cerebrospinal fluid (CSF) circulating tumour DNA (ctDNA) recapitulates the genomic alterations of the tumour and facilitates subgrouping and risk stratification, providing valuable information about diagnosis and prognosis. CSF ctDNA also characterises the intra-tumour genomic heterogeneity identifying small subclones. ctDNA is abundant in the CSF but barely present in plasma and longitudinal analysis of CSF ctDNA allows the study of minimal residual disease, genomic evolution and the characterisation of tumours at recurrence. Ultimately, CSF ctDNA analysis could facilitate the clinical management of medulloblastoma patients and help the design of tailored therapeutic strategies, increasing treatment efficacy while reducing excessive treatment to prevent long-term secondary effects.

Molecular profiling of tumors of the brainstem by sequencing of CSF-derived circulating tumor DNA.

Brainstem gliomas are molecularly heterogeneous diseases, many of which are difficult to safely surgically resect and have limited treatment options due to their eloquent location. These constraints pose challenges to biopsy, which limits the use of routine molecular profiling and identification of personalized therapies. Here, we explored the potential of sequencing of circulating tumor DNA (ctDNA) isolated from the cerebrospinal fluid (CSF) of brainstem glioma patients as a less invasive approach for tumor molecular profiling. CSF was obtained from patients either intraoperatively (91.2%, 52/57), from ventricular-peritoneal shunt (3.5%, 2/57), or by lumbar puncture (5.3%, 3/57), all prior to surgical manipulation of the tumor. Deep sequencing of glioma-associated genes was performed on CSF-derived ctDNA and, where available, matched blood and tumor DNA from 57 patients, including nine medullary and 23 diffuse intrinsic pontine gliomas (DIPG). At least one tumor-specific mutation was detected in over 82.5% of CSF ctDNA samples (47/57). In cases with primary tumors harboring at least one mutation, alterations were identified in the CSF ctDNA of 97.3% of cases (36/37). In over 83% (31/37) of cases, all primary tumor alterations were detected in the CSF, and in 91.9% (34/37) of cases, at least half of the alterations were identified. Among ten patients found to have primary tumors negative for mutations, 30% (3/10) had detectable somatic alterations in the CSF. Finally, mutation detection using plasma ctDNA was less sensitive than sequencing the CSF ctDNA (38% vs. 100%, respectively). Our study indicates that deep sequencing of CSF ctDNA is a reliable technique for detecting tumor-specific alterations in brainstem tumors. This approach may offer an alternative approach to stereotactic biopsy for molecular profiling of brainstem tumors.

Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases.

Challenges in obtaining tissue specimens from patients with brain tumours limit the diagnosis and molecular characterisation and impair the development of better therapeutic approaches. The analysis of cell-free tumour DNA in plasma (considered a liquid biopsy) has facilitated the characterisation of extra-cranial tumours. However, cell-free tumour DNA in plasma is limited in quantity and may not reliably capture the landscape of genomic alterations of brain tumours. Here, we review recent work assessing the relevance of cell-free tumour DNA from cerebrospinal fluid in the characterisation of brain cancer. We focus on the advances in the use of the cerebrospinal fluid as a source of cell-free tumour DNA to facilitate diagnosis, reveal actionable genomic alterations, monitor responses to therapy, and capture tumour heterogeneity in patients with primary brain tumours and brain and leptomeningeal metastases. Profiling cerebrospinal fluid cell-free tumour DNA provides the opportunity to precisely acquire and monitor genomic information in real time and guide precision therapies.

Detection of Histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma.

Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of "liquid biopsy" in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response.

Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid.

PURPOSE: Cancer spread to the central nervous system (CNS) often is diagnosed late and is unresponsive to therapy. Mechanisms of tumor dissemination and evolution within the CNS are largely unknown because of limited access to tumor tissue. MATERIALS AND METHODS: We sequenced 341 cancer-associated genes in cell-free DNA from cerebrospinal fluid (CSF) obtained through routine lumbar puncture in 53 patients with suspected or known CNS involvement by cancer. RESULTS: We detected high-confidence somatic alterations in 63% (20 of 32) of patients with CNS metastases of solid tumors, 50% (six of 12) of patients with primary brain tumors, and 0% (zero of nine) of patients without CNS involvement by cancer. Several patients with tumor progression in the CNS during therapy with inhibitors of oncogenic kinases harbored mutations in the kinase target or kinase bypass pathways. In patients with glioma, the most common malignant primary brain tumor in adults, examination of cell-free DNA uncovered patterns of tumor evolution, including temozolomide-associated mutations. CONCLUSION: The study shows that CSF harbors clinically relevant genomic alterations in patients with CNS cancers and should be considered for liquid biopsies to monitor tumor evolution in the CNS.

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.

Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma.

Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord.

Cell-free DNA shed by cancer cells has been shown to be a rich source of putative tumor-specific biomarkers. Because cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we studied whether the cerebrospinal fluid (CSF) that bathes the CNS is enriched for tumor DNA, here termed CSF-tDNA. We analyzed 35 primary CNS malignancies and found at least one mutation in each tumor using targeted or genome-wide sequencing. Using these patient-specific mutations as biomarkers, we identified detectable levels of CSF-tDNA in 74% [95% confidence interval (95% CI) = 57-88%] of cases. All medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space were detectable (100% of 21 cases; 95% CI = 88-100%), whereas no CSF-tDNA was detected in patients whose tumors were not directly adjacent to a CSF reservoir (P < 0.0001, Fisher's exact test). These results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.

Brain tumor mutations detected in cerebral spinal fluid.

BACKGROUND: Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood-brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative "liquid biopsy" of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined. METHODS: We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease. RESULTS: We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from <5 to nearly 3000 copies/mL CSF. We identified 7 somatic mutations from the CSF of a patient with leptomeningeal disease by use of cancer panel sequencing, and the result was concordant with genetic testing on the primary tumor biopsy. CONCLUSIONS: Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.

Methylation of the hTERT promoter: a novel cancer biomarker for leptomeningeal metastasis detection in cerebrospinal fluids.

PURPOSE: The diagnosis of leptomeningeal metastases is usually confirmed by the finding of malignant cells by cytologic examination in the cerebrospinal fluid (CSF). More sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Promoter methylation of the human telomerase reverse transcriptase (hTERT) gene characterizes most cancer cells. The aim of this study was to develop a sensitive method to detect hTERT methylation and to explore its use as a cancer biomarker in CSF. EXPERIMENTAL DESIGN: In 77 CSF specimens from 67 patients, hTERT promoter methylation was evaluated using real-time methylation-sensitive high-resolution melting (MS-HRM) and real-time TaqMan PCR and MS-HRM in a single-tube assay. RESULTS: Real-time MS-HRM assay was able to detect down to 1% hTERT-methylated DNA in a background of unmethylated DNA. PCR products were obtained from 90% (69/77) of CSF samples. No false positive hTERT was detected in the 21 non-neoplastic control cases, given to the method a specificity of 100%. The sensitivity of the real-time MS-HRM compared with the cytologic gold standard analysis was of 92% (11/12). Twenty-six CSFs from 22 patients with an hTERT-methylated primary tumor showed cytologic results suspicious for malignancy; in 17 (65%) of them, a diagnosis of leptomeningeal metastases could be confirmed by the hTERT methylation test. CONCLUSION: The hTERT real-time MS-HRM approach is fast, specific, sensitive, and could therefore become a valuable tool for diagnosis of leptomeningeal metastases as an adjunct to the traditional examination of CSF.

Prognostic value of free DNA quantification in serum and cerebrospinal fluid in glioma patients.

Unlike uniformly truncated DNA released from apoptotic nondiseased cells, free DNA released from dead tumor cells varies in size. Free DNA has been considered as a candidate biomarker for malignant tumors. We obtained serum samples from 70 patients with glioma and 22 healthy volunteers as control and cerebrospinal fluid (CSF) samples from 20 patients with glioma and eight nonneoplastic controls with hydrocephalus or arachnoid cyst and performed preoperative analysis of free DNA concentration and integrity by a quantitative polymerase chain reaction. With two primers sets amplifying short and long free DNA fragments (ALU115 and ALU247), free DNA integrity was determined by ratio of the concentration of ALU247 over ALU115 (ALU247/115). Our results indicate that free DNA integrity and the ratio of long fragments to short fragments may be a useful diagnostic assay for glioma. In summary, the CSF-free DNA concentration and integrity may serve as a new marker for the diagnosis of glioma.

Detection of epithelial growth factor receptor mutations in cerebrospinal fluid from patients with lung adenocarcinoma suspected of neoplastic meningitis.

BACKGROUND: Neoplastic meningitis (NM) is a devastating neurological complication of cancer that needs to be diagnosed in the early stages of disease. Polymerase chain reaction detection of epithelial growth factor receptor (EGFR) mutations in cerebrospinal fluid (CSF), which are predictive markers for EGFR tyrosine kinase inhibitor therapy in lung cancer, might be important to diagnose and to treat NM in patients with lung cancer. In this study, we attempted to detect EGFR mutations in CSF and to compare EGFR status between CSF and primary or metastatic lesions in patients with lung adenocarcinoma suspected of NM. METHODS: Twenty-nine patients with lung adenocarcinoma suspected of having NM underwent lumbar puncture. EGFR status of CSF was analyzed by direct DNA sequencing. EGFR mutations of primary or metastatic lesions (lymph nodes and bones) were analyzed in 20 cases. RESULTS: EGFR mutations were detected in CSF of 13 (45%) of 29 patients. In 5 (31%) of 16 patients with negative CSF cytology, EGFR mutations were detected. In four patients, EGFR mutations were shown in CSF, but not in primary or metastatic lesions. Conversely, in two patients, EGFR mutations were shown in primary or metastatic lesions, but not in CSF despite positive CSF cytology. CONCLUSIONS: EGFR mutations, suggesting the existence of malignant cells, were detected in CSF, even in patients with non-small cell lung cancer with negative cytological results. EGFR mutations in CSF do not always reflect the same status as in primary or metastatic lesions.

Detection of MYCN DNA in the cerebrospinal fluid for diagnosing isolated central nervous system relapse in neuroblastoma.

We present the case of a 1-year-old female with stage-4 neuroblastoma with MYCN amplification; she was treated with five chemotherapy courses, resulting in normalization of elevated serum levels of tumor markers. Complete remission was achieved after allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning. Nine months later, however, the tumor relapsed in the central nervous system (CNS). The serum and cerebrospinal fluid (CSF) levels of the tumor markers were normal, but the MYCN copy number was high only in the CSF DNA, suggesting an isolated CNS recurrence. The MYCN copy number in the CSF DNA was reflective of response to treatment.

[Low-molecular DNA in blood plasma and spinal fluid of cancer patients].

Enhanced preoperative levels of low-molecular DNA in blood plasma and in cerebrospinal fluid were established in cancer patients. They rose even higher after surgery due to stress. Possible mechanisms are discussed.

Significance of an epidermal growth factor receptor mutation in cerebrospinal fluid for carcinomatous meningitis.

We report a case of epidermal growth factor receptor (EGFR) inhibitor-sensitive lung adenocarcinoma with carcinomatous meningitis who showed a good response to gefitinib, an oral tyrosine kinase inhibitor of EGFR. This good response to gefitinib treatment was attributed to evidence of an EGFR mutation, L858R in exon 21, which was detected in a small amount of cerebrospinal fluid (CSF) before the positive CSF cytology. Patients with carcinomatous meningitis often have a poor performance status, and therefore diagnostic approaches and therapeutic methods are also often limited. Detection of EGFR mutations may be a useful method for non-small cell lung cancer diagnosis, and also facilitate determination of appropriate therapeutic protocols.

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Polymerase chain reaction on cerebrospinal fluid cells in suspected leptomeningeal involvement in childhood acute lymphoblastic leukemia: comparison to cytomorphological analysis.

The leptomeningeal involvement of central nervous system is defined in the most centers by the presence of blast cells in the CSF or the presence of cranial-nerve palsies. Sometimes, cytology does not allow clear distinction between lymphoblasts and normal cells, and auxiliary methods to the precise identification of leukemic cells in cerebrospinal fluid is necessary. We analyzed CSF from 11 consecutive patients, in whom a differential diagnosis of leptomeningeal involvement was made, including 4 patients at diagnosis and 7 patients during the treatment by cytomorphological analysis and PCR and automatic sequencing. Six patients were considered with leptomeningeal involvement by conventional analysis: unequivocal cytomorphological involvement was considered in 5 patients, and in one it was assumed to be due to cranial-nerve palsy, with no blast cells detected in cerebrospinal fluid. In 2 it was considered suspicious and in 3 negative. PCR and sequencing analysis showed involvement in 6 patients; 5 of the 6 patients were considered to have leptomeningeal involvement based on clinical and cytomorphological criteria, and, in one of the patients, it was suspicious. Our data suggest that the use of PCR and sequencing can be useful in confirming CNS leukemia and eliminating other conditions when used together with the cytomorphological analysis.