Cerebrospinal Fluid Cell-free DNA Research Articles

Sequencing of cerebrospinal fluid cell-free DNA facilitated early differential diagnosis of intramedullary spinal cord tumors

Pre-surgery differential diagnosis is valuable for personalized treatment planning in intramedullary spinal cord tumors. This study assessed the performance of sequencing cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) for differential diagnosis of these tumors. Prospectively enrolling 45 patients with intramedullary spinal cord lesions, including diffuse midline glioma (DMG), H3K27-altered (14/45), glioblastoma (1/45), H3-wildtype-astrocytoma (10/45), ependymoma (11/45), and other lesions (9/45), CSF samples were collected via lumbar puncture (41/45), intraoperative extraction (3/45), and Ommaya reservoir (1/45). Then, these samples underwent targeted sequencing along with paired tissue DNA. DMG, H3K27-altered patients exhibited a higher ctDNA positivity (85.7%, 12/14) compared to patients with H3-wildtype-astrocytoma (0/8, P = 0.0003), ependymoma (2/10, P = 0.003), and glioneuronal tumor (0/3, P = 0.009). The histological-grade-IV (P = 0.0027), Ki-67 index ≥10% (P = 0.014), and tumor reaching spinal cord surface (P = 0.012) are also associated with higher ctDNA positivity. Interestingly, for patients with TERT promoter mutant tumors, TERT mutation was detectable in the CSF cfDNA of one DMG case, but not other five cases with histological-grade-II tumors. Shared copy number variants were exclusively observed in DMG, H3K27-altered, and showed a strong correlation (Correlation = 0.95) between CSF and tissue. Finally, H3K27M mutations in CSF exhibited high diagnostic efficiency for DMG, H3K27-altered (Sensitivity = 85.7%, Specificity = 100.0%, AUC = 0.929). Notably, H3K27M was detectable in CSF from patients with recurrent tumors, making it easily applicable for postoperative monitoring. In conclusion, the molecular profile from ctDNA released into CSF of malignant tumors was more frequently detected compared to relatively benign ones. Sequencing of ctDNA in CSF exhibited high efficiency for the differential diagnosis of DMG, H3K27-altered.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer.

e16022 Background: Leptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was under-investigated in GCLM. Methods: We retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes. Results: CSF had higher mutation allele frequency ( P=0.015), more somatic mutations ( P=0.032), and more copy-number variations ( P<0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival ( P=0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation ( P=0.014), IGF1R mutation ( P=0.034), AR mutation ( P=0.038), SMARCB1 deletion ( P<0.001), SMAD4 deletion ( P=0.0034), and TGF-beta pathway aberration ( P=0.0038). Additionally, improvement in intracranial pressure ( P<0.001), improvement in CSF cytology ( P=0.0038), and relatively low levels of CSF ctDNA ( P=0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment. Conclusions: CSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer

BackgroundLeptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was underinvestigated in GCLM.MethodsWe retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes.ResultsCSF had higher mutation allele frequency (P = 0.015), more somatic mutations (P = 0.032), and more copy-number variations (P < 0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival (P = 0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation (P = 0.014), IGF1R mutation (P = 0.034), AR mutation (P = 0.038), SMARCB1 deletion (P < 0.001), SMAD4 deletion (P = 0.0034), and TGF-beta pathway aberration (P = 0.0038). Additionally, improvement in intracranial pressure (P < 0.001), improvement in CSF cytology (P = 0.0038), and relatively low levels of CSF ctDNA (P = 0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment.ConclusionsCSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Cell-Free DNA in Cerebrospinal Fluid Complements the Monitoring Value of Interleukin-10 in Newly Diagnosed Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) usually has a poor prognosis. Cerebrospinal fluid (CSF) interleukin (IL)-10 has shown diagnostic, prognostic, and monitoring value in our previous studies. Cell-free circulating tumor DNA can be detected in the CSF of refractory/relapse cases and has also shown monitoring value. However, information about its monitoring value in newly diagnosed PCNSL patients and comparisons of CSF IL-10 and CSF cell-free DNA (cfDNA) are scarce. We performed next-generation sequencing on paraffin-embedded tissue and the serial CSF cfDNA of 10 newly diagnosed PCNSL patients and on the baseline CSF cfDNA of 11 other central nervous system lymphoma patients. We also monitored the CSF IL-10 levels of the 10 newly diagnosed PCNSL patients. In seven newly diagnosed PCNSL patients with sufficient baseline CSF cfDNA, six had ≥1 mutated genes in their CSF cfDNA. The most common were MYD88(4/7), PIM1(3/7), MLL2(3/7), and ETV6(2/7). We also identified multiple somatic mutations, most commonly in PIM1. MYD88L265P can be detected in both tumor tissue and CSF cfDNA. The genomic profiles of CFS cfDNA were similar in PCNSL and PIOL patients. Newly diagnosed PCNSL patients with persistently positive cfDNA and negative IL-10 progressed quickly, while those with negative cfDNA and negative IL-10 were in maintenance therapy for more than 18 months. Two patients without cfDNA had increased CSF IL-10 concentrations before disease relapse. These results indicate that negative CSF cfDNA predicts better results, and persistently positive CSF cfDNA predicts disease progression earlier than conventional magnetic resonance imaging. In conclusion, CSF cfDNA is a potential predictor of relapse and progression, which complements the monitoring value of CSF IL-10 in newly diagnosed PCNSL patients.

Genomic Instability in Cerebrospinal Fluid Cell-Free DNA Predicts Poor Prognosis in Solid Tumor Patients with Meningeal Metastasis.

Simple SummaryWe established a genomic instability score using unfiltered sequencing data from meningeal metastasis (MM) cell-free circulating tumor DNA (ctDNA) samples and found that substantial genomic instability (GI) was present in cerebrospinal fluid ctDNA rather than plasma ctDNA, implying that MM lesions have a significantly increased GI status compared to primary tumors or extracranial metastatic lesions, which may suggest tumor clonal evolution. We also found that high GI status was an independent poor prognostic factor in lung adenocarcinoma MM patients, including meningeal metastasis-free survival (MFS) and overall survival (OS). Considering that genomically unstable tumors are more sensitive to PARP inhibitors, targeting GI alone or in combination with conventional therapy may be a promising treatment strategy for solid tumor patients with MM.Genomic instability (GI), which leads to the accumulation of DNA loss, gain, and rearrangement, is a hallmark of many cancers such as lung cancer, breast cancer, and colon cancer. However, the clinical significance of GI has not been systematically studied in the meningeal metastasis (MM) of solid tumors. Here, we collected both cerebrospinal fluid (CSF) and plasma samples from 56 solid tumor MM patients and isolated cell-free ctDNA to investigate the GI status using a next-generation sequencing-based comprehensive genomic profiling of 543 cancer-related genes. According to the unfiltered heterozygous mutation data-derived GI score, we found that 37 (66.1%) cases of CSF and 3 cases (6%) of plasma had a high GI status, which was further validated by low-depth whole-genome sequencing analysis. It is demonstrated that a high GI status in CSF was associated with poor prognosis, high intracranial pressure, and low Karnofsky performance status scores. More notably, a high GI status was an independent poor prognostic factor of poor MM-free survival and overall survival in lung adenocarcinoma MM patients. Furthermore, high occurrences of the co-mutation of TP53/EGFR, TP53/RB1, TP53/ERBB2, and TP53/KMT2C were found in MM patients with a high GI status. In summary, the GI status in CSF ctDNA might be a valuable prognostic indicator in solid tumor patients with MM.

Diagnosis of Leptomeningeal Metastasis in Women With Breast Cancer Through Identification of Tumor Cells in Cerebrospinal Fluid Using the CNSide™ Assay

Diagnosis of LM is limited by low sensitivity of cerebrospinal fluid (CSF) cytopathology. Detecting tumor cells in CSF (CSF-TCs) might be more sensitive. We evaluated if CNSide (CNSide), a novel assay for tumor cell detection in CSF, can detect CSF-TCs better than conventional CSF cytology. We enrolled adults with metastatic breast cancer and clinical suspicion for LM to undergo lumbar puncture (LP) for CSF cytopathology and CNSide. CNSide captured CSF-TCs using a primary 10-antibody mixture, streptavidin-coated microfluidic channel, and biotinylated secondary antibodies. CSF-TCs were assessed for estrogen receptor (ER) expression by fluorescent antibody and HER2 amplification by fluorescent in situ hybridization (FISH). CSF cell-free DNA (cfDNA) was extracted for next-generation sequencing (NGS). Leptomeningeal disease was defined as positive CSF cytology and/or unequivocal MRI findings. We calculated sensitivity and specificity of CSF cytology and CNSide for the diagnosis of LM. Ten patients, median age 51 years (range, 37-64), underwent diagnostic LP with CSF evaluation by cytology and CNSide. CNSide had sensitivity of 100% (95% Confidence Interval [CI], 40%-100%) and specificity of 83% (95% CI, 36%-100%) for LM. Among these patients, concordance of ER and HER2 status between CSF-TCs and metastatic biopsy were 60% and 75%, respectively. NGS of CSF cfDNAidentified somatic mutations in three patients, including one with PIK3CA p.H1047L in blood and CSF. CNSide may be a viable platform to detect CSF-TCs, with potential use as a diagnostic tool for LM in patients with metastatic breast cancer. Additional, larger studies are warranted.

WITHDRAWN: Unique genomic alterations of cerebrospinal fluid cell-free DNA are critical for targeted therapy of non-small cell lung cancer with leptomeningeal metastasis

We reported unique molecular features of cerebrospinal fluid (CSF) of NSCLC patients with leptomeningeal metastasis (LM), suggesting to establish CSF as a better liquid biopsy in clinical practices. We performed next-generation panel sequencing of primary tumor tissue, plasma and CSF from 131 NSCLC patients with LM, and observed high somatic copy number variations (CNV) in CSF of NSCLC patients with LM. The status of EGFR-activating mutations was highly concordant between CSF, plasma, and primary tumors. ALK translocation was detected in 8.3% of tumor tissues, but only 2.4% in CSF and 2.7% in plasma. Others such as ROS1 rearrangement, RET fusion, HER2 mutation, NTRK1 fusion, and BRAF V600E mutation were detected in 7.9% of CSF and 11.1% of tumor tissues, but only 4% in plasma. Our study has shed light on the unique genomic variations of CSF and demonstrated that CSF may represent better liquid biopsy for NSCLC patients with LM.

Unique Genomic Alterations of Cerebrospinal Fluid Cell-Free DNA Are Critical for Targeted Therapy of Non-Small Cell Lung Cancer With Leptomeningeal Metastasis.

We reported unique molecular features of cerebrospinal fluid (CSF) of nonsmall cell lung cancer (NSCLC) patients with leptomeningeal metastasis (LM), suggesting establishing CSF as a better liquid biopsy in clinical practices. We performed next-generation panel sequencing of primary tumor tissue, plasma, and CSF from 131 NSCLC patients with LM and observed high somatic copy number variations (CNV) in CSF of NSCLC patients with LM. The status of EGFR-activating mutations was highly concordant between CSF, plasma, and primary tumors. ALK translocation was detected in 8.3% of tumor tissues but only 2.4% in CSF and 2.7% in plasma. Others such as ROS1 rearrangement, RET fusion, HER2 mutation, NTRK1 fusion, and BRAF V600E mutation were detected in 7.9% of CSF and 11.1% of tumor tissues but only 4% in plasma. Our study has shed light on the unique genomic variations of CSF and demonstrated that CSF might represent better liquid biopsy for NSCLC patients with LM.

Utility of Cerebrospinal Fluid Cell-Free DNA in Patients with EGFR-Mutant Non-Small-Cell Lung Cancer with Leptomeningeal Metastasis.

Leptomeningeal metastasis (LM) is a fatal complication of advanced non-small-cell lung cancer (NSCLC). The aim of this study was to evaluate the utility of cerebrospinal fluid (CSF) as a medium for epidermal growth factor receptor (EGFR) mutation testing in clinical practice. We prospectively enrolled patients with EGFR-mutant NSCLC who underwent CSF sampling for suspected LM. The supernatant of CSF after routine cytology examination was collected. The diagnosis of LM was established according to EANO-ESMO criteria. CSF and plasma cell-free DNA (cfDNA) were retrieved for EGFR mutation testing. Fifty-one patients with a median age of 62.7 years were enrolled. The median duration from initial diagnosis to CSF sampling was 23.0 months and most patients (94.1%) had received at least one EGFR-tyrosine kinase inhibitor. Adenocarcinoma cells were found in 37 CSF samples (72.5%), and 48 (94.1%) patients had confirmed or probable LM. Thirty-five of these 48 patients (72.9%) had valid EGFR mutation-testing results using CSF cfDNA and tended to have higher white blood cell counts and positive cytology in their CSF compared to those with invalid mutation testing results. The overall detection rate of EGFR mutation in CSF cfDNA was 68.8%, and the T790M detection rate was 14.6%. In 37 patients with paired CSF and plasma samples, the concordance rate of the EGFR mutation results was 29.7%. For patients with EGFR-mutant NSCLC with LM, CSF supernatant is a valuable source for EGFR mutation testing and may provide important information.

Cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain.

Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.

A nanowire-based liquid biopsy method using cerebrospinal fluid cell-free DNA for targeted management of leptomeningeal carcinomatosis.

This study aimed to evaluate whether genotyping cell free DNA (cfDNA) in the cerebrospinal fluid (CSF) may be helpful in managing leptomeningeal carcinomatosis (LMC) of EGFR-mutant non-small cell lung cancer (NSCLC). Patients with EGFR-mutant NSCLC who progressed as LMC after 3rd-generation tyrosine kinase inhibitors (EGFR-TKIs) were evaluated. A nanowire-based cfDNA assay was performed for genotyping cfDNA from the CSF and plasma. We focused on de novo EGFR C797S mutation and MET amplification, which are the most common mechanisms of resistance to 3rd-generation EGFR-TKIs. Among 11 patients, five (45.5%) had progression only at the leptomeninges. The tumor-associated CSF-cfDNA was identified in eight (72.7%) patients, and plasma-cfDNA in six (54.5%) patients. In the CSF-cfDNA, EGFR C797S mutation and MET amplification were detected in four (36.3%) and two (18.2%) patients, respectively. Of four patients with the C797S-positive LMC, only one had concurrent CSF-T790M mutation. Three patients who had the C797S-positive LMC without CSF-T790M mutation, received 1st-2nd generation EGFR-TKIs and showed clinical benefits for 20.8, 17.8, and 8.8weeks, respectively. Serial assessment with cfDNA in these patients demonstrated that the CSF levels of C797S mutation were decreased with radiological or neurological improvement but the plasma levels of T790M mutation were markedly increased before objective progression. Genotyping CSF-cfDNA by the nanowire-based assay is feasible and effective in guiding the treatment of LMC in patients with EGFR-mutant NSCLC.

Cerebrospinal Fluid Cell Free DNA Demonstrates Unique Genomic Alterations in Non-Small Cell Lung Cancer with Leptomeningeal Metastasis

Background: We retrospectively reported unique molecular features of cerebrospinal fluid(CSF) of NSCLC patients with LM, suggesting to establish CSF as a better liquid biopsy method in clinical practices. Methods: We performed next-generation panel sequencing of primary tumor tissue, plasma and cerebrospinal fluid (CSF) from 131 NSCLC patients with LM. Findings: High somatic copy number variations (CNV) were observed in CSF compared with plasma and primary tumor, exhibiting unique molecular features of CSF of NSCLC patients with LM. The status of EGFR-activating mutations was highly concordant between CSF, plasma, and primary tumors. There was no statistical difference between CSF, plasma and tumor tissues to detect EGFR mutation. ALK translocation was detected in 8.3% of tumor tissues, but detection rate was only 2.4% in CSF and 2.7% in plasma. Except EGFR and ALK, other rare driver genes such as ROS1 rearrangement, RET fusion, HER2 mutation, NTRK1 fusion, and BRAF V600E mutation were detected in 7.9% of CSF and 11.1% of tumor tissues, but only 4% in plasma. Except EGFR, TP53 and RB1 aberrations were high in these patients with CSF and primary tumor samples, implicating a prediction of high LM risk. Interestingly, co-occurring of EGFR and TP53 was not different among CSF, plasma and tumor tissues. However, co-occurring of EGFR and RB1 and EGFR/TP53/RB1 were higher in CSF compared with plasma or tumor tissues. Interpretation: Our study has shed light on the unique genomic variations of CSF and demonstrated that CSF may represent better liquid biopsy for NSCLC patients with LM. Funding Statement: This study was funded by the grants from the National Young 1000 Talents Program of China and Jiangsu Province Education Department, Jiangsu Province “Innovative and Entrepreneurial Team” and “Innovative and Entrepreneurial Talent.” Declaration of Interests: The authors stated: None. Ethics Approval Statement: All patients provided signed informed consent and the study protocol was approved by the Research Ethics Committee of Nanjing Drum Tower Hospital.

Circulating Tumor DNA Analysis for Patients with Oncogene-Addicted NSCLC With Isolated Central Nervous System Progression.

In patients with oncogene-addicted NSCLC and isolated central nervous system progression (iCNS), tissue biopsy is challenging, and the clinical utility of plasma liquid biopsy (i.e., circulating tumor DNA [ctDNA]) is unknown. Patients with advanced NSCLC with known baseline genomic alteration (GA) (EGFR, ALK, BRAF, KRAS, HER2, ROS1, MET, PIK3CA, STK11, TP53) on tissue were divided into three groups on the basis of their disease progression pattern: iCNS, extra-CNS only (noCNS), or both (cCNS). All patients with available plasma ctDNA were included and were analyzed by next-generation sequencing InVisionFirst-Lung. ctDNA was considered positive if at least one GA was detected. Cell-free tumor DNA was analyzed in cerebrospinal fluid when available. Out of 517 patients screened, 247 were included: 54 had iCNS, 99 had noCNS, and 94 had cCNS progressive disease (64, 128, and 110 ctDNA samples, respectively). CtDNA was positive in 52% iCNS versus 84% in noCNS and 92% in cCNS (p < 0.00001), with lower detection of driver (37% versus 77% and 73%, respectively) and resistance alterations (6% versus 45% and 44%). Patients with iCNS and positive ctDNA were more at risk of extra-CNS progression (32% versus 7%, p= 0.026). In 12 patients with iCNS, ctDNA was positive in six (50%) plasma and in 10 (83%) paired cerebrospinal fluid (p= 0.193). Although tagged amplicon-based next-generation sequencing has high detection rates of GA in plasma ctDNA in patients with NSCLC with extra-CNS disease, detection rate of GAs (52%) is lower in the subset of patients with iCNS disease. Complementary tests such as cerebrospinal fluid cell-free DNA may be useful. Further evidence would be beneficial to understand the genomic landscape in patients with NSCLC and iCNS.

Clinical Utility of Cerebrospinal Fluid Cell-Free DNA as Liquid Biopsy for Leptomeningeal Metastases in ALK-Rearranged NSCLC

IntroductionLeptomeningeal metastases (LMs) indicated a poor prognosis in NSCLC. LMs were more frequent in driver gene–mutated patients, and cerebrospinal fluid (CSF) cell-free DNA has shown unique genetic profiles of LM in EGFR-mutated LM. However, studies in patients with ALK receptor tyrosine kinase gene (ALK)-rearranged NSCLC with LMs are scarce. MethodsPatients with lung cancer with ALK rearrangement were screened from September 2011 to February 2018 at our institute. CSF and paired plasma were tested by next-generation sequencing. ResultsLMs were diagnosed in 30 (10.3%) of 291 patients with ALK-rearranged lung cancer. A total of 11 paired CSF and plasma samples tested by next-generation sequencing were analyzed. Driver genes were detected in 81.8% of the CSF samples (9 of 11) and 45.5% of the plasma samples (5 of 11) (p = 0.183). The maximum allelic fractions were all higher in CSF than in plasma (p = 0.009). ALK and tumor protein p53 gene (TP53) were the two most frequently mutated genes in CSF. Gatekeeper gene ALK G1202R and C1156F mutations were identified in CSF after resistance to alectinib. Multiple copy number variants were mainly found in CSF, including in EGFR, cyclin D1 gene (CCND1), fibroblast growth factor 3 gene (FGF3), and fibroblast growth factor 4 gene (FGF4). Also found were v-myc avian myelocytomatosis viral oncogene homolog gene (MYC) copy number gains and TP53 and cyclin dependent kinase inhibitor 2A gene (CDKN2A) copy number deletions. Brigatinib seemed to be effective in controlling LM. One case showed that CSF could be used to monitor disease development of LM and longitudinally monitor tumor response. ConclusionLiquid biopsy of CSF is more sensitive than liquid biopsy of plasma to detect targetable alterations, characterizing resistance mechanisms on progression and monitoring tumor response in patients with ALK-rearranged NSCLC with LM. Thus, CSF might be promising as a medium of liquid biopsy in LM.

Unique genomic profiles obtained from cerebrospinal fluid cell-free DNA of non-small cell lung cancer patients with leptomeningeal metastases

ABSTRACTBackground: Leptomeningeal metastases (LM), associated with poor prognosis, are frequent complications of advanced non-small cell lung cancer (NSCLC) patients, especially in patients with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the mutational landscape of LM has not been comprehensively investigated in large cohorts and the underlining biology of LM remains elusive. Some studies have explored the potential of cerebrospinal fluid (CSF) in reflecting the molecular profile of LM but with limited number of patients enrolled.Methods: In this study, we performed capture-based targeted sequencing using a panel consisting of 168 lung cancer-related genes on matched CSF and plasma samples from 72 advanced NSCLC patients with confirmed LM to interrogate the potential of CSF as a source of liquid biopsy.Results: We revealed a rate of detection of 81.5% and 62.5% for CSF and plasma, respectively (p = 0.008). The maximum allelic fraction (MaxAF) was also significantly higher in CSF (43.6% vs. 4.6%) (p < 0.001). CSF, harboring a unique genomic profile by having a significant number of CSF-specific mutations, primarily copy number variations, is superior to plasma in reflecting the mutational profile of LM. Further pathway enrichment analysis revealed that most of CSF-specific mutations participated in pathways relevant to the tumorigenesis and the development of metastases. Moreover, our data also revealed that TP53 loss of heterozygosity (LOH) predominantly existed in CSF (p < 0.001).Conclusions: Collectively, we demonstrated that CSF provides a more comprehensive profile of LM than plasma in a large cohort, thus can be used as an alternative source of liquid biopsy for LM patients.

MA16.10 Clinical Utility of Cerebrospinal Fluid Cell-Free DNA for Clarifying Genetic Features of Leptomeningeal Metastases in ALK Rearrangement NSCLC

MA16.10 Clinical Utility of Cerebrospinal Fluid Cell-Free DNA for Clarifying Genetic Features of Leptomeningeal Metastases in ALK Rearrangement NSCLC

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: a new medium of liquid biopsy

BackgroundLeptomeningeal metastases (LM) are more frequent in non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the purpose of this study was to explore the potential role of cerebrospinal fluid (CSF) as a source of liquid biopsy in patients with LM. Patients and methodsPrimary tumor, CSF, and plasma in NSCLC with LM were tested by next-generation sequencing. In total, 45 patients with suspected LM underwent lumbar puncture, and those with EGFR mutations diagnosed with LM were enrolled. ResultsA total of 28 patients were enrolled in this cohort; CSF and plasma were available in 26 patients, respectively. Driver genes were detected in 100% (26/26), 84.6% (22/26), and 73.1% (19/26) of samples comprising CSF cell-free DNA (cfDNA), CSF precipitates, and plasma, respectively; 92.3% (24/26) of patients had much higher allele fractions in CSF cfDNA than the other two media. Unique genetic profiles were captured in CSF cfDNA compared with those in plasma and primary tissue. Multiple copy number variations (CNVs) were mainly identified in CSF cfDNA, and MET copy number gain identified in 47.8% (11/23) of patients was the most frequent one, while other CNVs included ERBB2, KRAS, ALK, and MYC. Moreover, loss of heterozygosity (LOH) of TP53 was identified in 73.1% (19/26) CSF cfDNA, which was much higher than that in plasma (2/26, 7.7%; P<0.001). There was a trend towards a higher frequency of concomitant resistance mutations in patients with TP53 LOH than those without (70.6% versus 33.3%; P=0.162). EGFR T790M was identified in CSF cfDNA of 30.4% (7/23) of patients who experienced TKI progression. ConclusionCSF cfDNA could reveal the unique genetic profiles of LM and should be considered as the most representative liquid biopsy medium for LM in EGFR-mutant NSCLC.

Detection of tumor-derived DNA mutations in cerebrospinal fluid of patients with primary or metastatic brain tumors.

2070 Background: Because detecting tumor-derived cell free DNA (cfDNA) in the blood of patients with primary or metastatic brain tumors is challenging, here we studied whether cerebrospinal fluid (CSF) could be serve as an alternative “liquid biopsy” by enabling measurement of circulating DNA within CSF to characterize tumor specific mutations. Methods: The paired cfDNA in CSF and plasma were collected from 20 patients with brain tumors and was subjected to enrichment for a 1.15M size panel cover exon regions from 1,086 genes. Followed by next generation sequencing on an Illumina X10 platform, the captured sequencing data was further processed using bioinformatics analysis to identify somatic mutations, including single nucleotide variants (SNV) and short insertions/deletions (indels). Results: The mutation profiles of 48 tumor associated genes in cfDNA were compared between the CSF and plasma. Our results showed that both average somatic mutation number and frequency identified in the cerebrospinal fluid was much higher than that in the corresponding plasma samples (25 vs. 18 &amp; 1.39% vs. 0.55%). Among the twenty cases, one more potential actionable mutation, EGFR exon 19 deletion mutation with a 25.38% allele frequency variation, was only detected in the CSF cfDNA of a patient with brain metastasis lung cancer. Conclusions: Tumor mutations were detectable in CSF cfDNA of patients with different primary and metastatic brain tumors. Thus cerebrospinal fluid cell free DNA analysis could be a potential alternative analysis for patients with primary or metastatic brain tumors.