Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids

Abstract

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.

Cerebrospinal fluid approach on neuro-oncology

“…These biomarkers may be nonspecific, such as β-glucuronidase, lactate dehydrogenase, beta2-microglobulin, carcinoembryonic antigen or alternatively. CSF release of tumor biomarkers markers has been demonstrated in many patients with LM, however, there was no clear correlation with the type of carcinoma or response to treatment observed 12,13,14 . Particular organ-specific tumor markers such as CA 15-3, CA 125, CA 19-9, CA724, AFP, NSE, Cyfra 21-1, EGFR, a-fetoprotein, b-human chorionic gonadotropin can be relatively specific for LM when elevated in CSF in the absence of markedly elevated serum levels 6,[11][12][13][14] .…”

“…CSF release of tumor biomarkers markers has been demonstrated in many patients with LM, however, there was no clear correlation with the type of carcinoma or response to treatment observed 12,13,14 . Particular organ-specific tumor markers such as CA 15-3, CA 125, CA 19-9, CA724, AFP, NSE, Cyfra 21-1, EGFR, a-fetoprotein, b-human chorionic gonadotropin can be relatively specific for LM when elevated in CSF in the absence of markedly elevated serum levels 6,[11][12][13][14] . Nonspecific tumor markers such as molecules involved in CNS penetration (eg, matrix metalloproteinases and cathepsins), tumor cell tropism (eg, chemokines CXCL8 and CCL18) and vascular endothelial growth factor can be strong indirect indicators of LM but none are sensitive enough to improve the cytological diagnosis 3,6,11,17 .…”

“…Biochemical markers, immunohistochemistry and molecular biology techniques applied to CSF have been explored in an attempt to find a reliable biological marker of disease 3,6,11 . Numerous biochemical markers have been evaluated, for early diagnosis and to effectiveness of treatment evaluation [12][13][14] . In general, their use has been limited by poor sensitivity and specificity.…”

“…Although serum levels are not able to distinguish systemic versus CNS involvement, CSF β2-microglobulin has been correlated with the presence of CNS disease in leukemia and lymphoma in small numbers of patients, as well as response to therapy. Levels of CSF β2-microglobulin may be elevated up to 50% in patients with leukemia or lymphoma and CSF involvement 14,16 . Malignant brain tumors may show an increased fraction of anaerobic LDH concentrations (LD4 and LD5) in CSF 16 .…”

“…Malignant brain tumors may show an increased fraction of anaerobic LDH concentrations (LD4 and LD5) in CSF 16 . When added to the determination of cell count and protein, oligoclonal bands testing contributes to the sensitivity of CSF analysis in diagnosing paraneoplastic syndromes 14,15 . Cerebrospinal fluid flow cytometry is a useful adjunct to CSF cytology and has been shown to increase the ability to detect CNS involvement in high-risk individuals, particularly in hematologic tumors 9,15 .…”