Cerebrospinal Fluid ctDNA Research Articles

Cerebrospinal fluid as a liquid biopsy for molecular characterization of brain metastasis in patients with non-small cell lung cancer

ObjectivesNon-small cell lung cancer (NSCLC) with brain metastases (BM) is a challenging clinical issue with poor prognosis. No data exist regarding extensive genetic analysis of cerebrospinal fluid (CSF) and its correlation to associated tumor compartments. Materials and MethodsWe designed a study across multiple NSCLC patients with matched material from four compartments; primary tumor, BM, plasma and CSF. We performed enrichment-based targeted next-generation sequencing analysis of ctDNA and exosomal RNA in CSF and plasma and compared the outcome with the solid tumor compartments. ResultsAn average of 105 million reads per sample was generated with fractions of mapped reads exceeding 99% in all samples and with a mean coverage above 10,000x. We observed a high degree of overlap in variants between primary lung tumor and BM. Variants specific for the BM/CSF compartment included in-frame deletions in AR, FGF10 and TSC1 and missense mutations in HNF1a, CD79B, BCL2, MYC, TSC2, TET2, NRG1, MSH3, NOTCH3, VHL and EGFR. ConclusionOur approach of combining ctDNA and exosomal RNA analyses in CSF presents a potential surrogate for BM biopsy. The specific variants that were only observed in the CNS compartments could serve as targets for individually tailored therapies in NSCLC patients with BM.

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 profiling of cerebrospinal fluid ctDNA to characterize molecular features and monitor intracranial responses of patients with osimertinib-resistant non-small cell lung cancer with leptomeningeal metastases.

e21095 Background: Leptomeningeal metastasis (LM) indicates an unfavorable prognosis in non-small cell lung cancer (NSCLC). Due to the blood-brain barrier, plasma is not an ideal medium to evaluate osimertinib resistance mechanisms in patients with LM. This study aimed to use cerebrospinal fluid (CSF) as a source of ctDNA for characterizing molecular features and monitoring intracranial responses from patients with leptomeningeal metastases who progressed on osimertinib. Methods: From Jan 2020 to Nov 2022, 65 patients with advanced NSCLC, including 32 osimertinib-treated patients, 6 other TKI-treated patients, and 27 treatment-naïve patients, in Beijing Tiantan Hospital were enrolled in this study. CSF obtained via lumbar puncture was used for targeted sequencing and cytologic analysis. In parallel, paired plasma from 35 patients was also subjected to targeted sequencing. Results: Among 65 patients, the overall positive rate of CSF ctDNA was 77% (50/65), 94% (34/36) in patients with LM, and 55% (16/29) in patients with brain parenchymal metastases (p = 0.0002). CSF ctDNA analysis had a higher consistency with the cytologic diagnosis of LM than imaging. Among the patients cytologic diagnosed with LM, 94% (32/34) of the patients had positive CSF ctDNA analysis at the same time, while the positive diagnostic rate of imaging was only 39% (13/33). EGFR alteration was found in 97% (29/30) CSF of patients with LM who progressed on Osimertinib, including L858R (67%), exon19 deletion (17%), T790M (10%), L861Q (7%), G719X (7%) and L747X (7%). Other recurrent genes discovered in the CSF were TP53 (73%), PIK3CA (43%), ERBB2 (33%), POLE (33%), ALK (27%), BRCA2 (27%), BRAF (23%), BRCA1 (23%), RB1 (20%) and PALB (20%). In the 35 paired CSF-plasma pairs, more alterations were uniquely found in CSF than in plasma (71% vs 17%), and only 12% of alterations were shared. Serial surveillance of CSF ctDNA in 7 cases of LM patients treated with osimertinib found that the VAF of CSF ctDNA showed a highly consistent trend with the tumor cell fraction of cytology. Conclusions: For NSCLC patients with LM, liquid biopsy of CSF is more sensitive than plasma to characterize osimertinib-resistant mechanisms and guide follow-up treatment. Serial surveillance of CSF ctDNA dynamics may reflect the intracranial tumor responses, which still needs to be validated in a large sample size.

Cerebrospinal fluid circulating tumor DNA for the detection and characterization of leptomeningeal metastasis in non-small cell lung cancer.

e21039 Background: Cerebrospinal fluid (CSF) has revealed the unique genetic characteristics of leptomeningeal metastasis (LM) from non-small cell lung cancer (NSCLC). However, the research in this area is still very limited. Methods: Patients with LM from NSCLC were retrospectively analyzed. Circulating tumor DNA (ctDNA) of CSFand pairedextracranial tissue or plasma were tested by ARMS or next-generation sequencing(NGS).Clinical outcomes were compared with Kaplan-Meier log-rank test and Coxproportional hazards methodologies. Results: We reviewed charts of 80 patients with LM treated atFujian Cancer Hospital. A total of 57 CSF and of which 30 contemporaneouslypaired extracranial samples tested by NGS were analyzed. Most interestingly, CSF ctDNA revealed EGFR mutations in 4 patients previously diagnosed as wild-type by extracranial specimens, and survival was extended by targeted therapy. In the gene map of paired samples, CSF showed more abundant cell-cycle regulatory genes and complex independent evolutionary. CDKN2A/2B CNVs-del was enriched threefold in CSF (~26.3%) with LM compared to extracranial specimens (~8.9%)in lung adenocarcinoma without LM. The median overall survival (OS) was 14.1 months [95% confidence interval (CI): 10.7–17.5 months]. Univariate analyses and multivariate Cox analyses demonstrated that patients with CDKN2A/CDKN2Bco-mutation had poor prognosis. Conclusions: CSF-based ctDNA testing contributes to the phenotypic detection and unique characterization of resistance mechanisms of LM in NSCLC. CDKN2A/2B mutation maybe a poor prognostic factor in LM patients.

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.

Abstract 1053: Circulating tumor DNA from cerebrospinal fluid (CSF) allows for characterization and monitoring of glioma patients

Abstract Intro: High-Grade Gliomas (HGGs) are the most common and aggressive primary brain tumors in adults and are almost always fatal. Liquid biopsy provides a noninvasive window into the cancer genome and the underlying biology of the tumor. Circulating-tumor DNA (ctDNA) is a versatile analyte for tumor diagnosis, monitoring treatment response, detecting resistance, and tracking tumor evolution. The central hypothesis of our work is that detection of cerebrospinal fluid (CSF) ctDNA correlates with clinically significant events and can be used as a surrogate for tissue biopsy to guide treatment decisions in the clinic. Methods: Our study includes CSF ctDNA samples from 140 recurrent glioma patients at Memorial Sloan Kettering Cancer Center who underwent collection of CSF as part of their clinical evaluation for neurological signs and symptoms. For each patient, glioma subtype and grade were confirmed by a neuropathologist. Overall survival was calculated as the time interval between the date of diagnosis and the date of death. All samples were sequenced using the MSK-IMPACT targeted sequencing assay (468 genes). Allele-specific copy number calls were assessed using the FACETS algorithm. Mutations were classified as drivers based on OncoKB. To evaluate the relationship between CSF ctDNA detection and clinico-pathologic correlates, CSF ctDNA status was determined positive by the presence of at least one somatic mutation and CSF ctDNA status was correlated with clinico-pathologic features. Results: Within this cohort, we found 68 CSF ctDNA positive and 72 CSF negative samples. The most frequently mutated genes were: TERT (58.2%), TP53 (47.8%), IDH1 (20.9%) and EGFR (26.9%). We observed a concordance between contemporaneously sampled tumor and CSF. In a multivariate analysis accounting for established prognostic factors including: % extent of resection at diagnosis; tumor burden at the time of lumbar puncture; and IDH status, we found that CSF ctDNA positivity was negatively correlated with overall survival (HR: 2.52, p &amp;lt;0.0001). Patients with CSF positive samples had an overall survival of 3.35 months vs 11.91 months for those with CSF negative samples (p &amp;lt; 0.0001). Conclusion: ctDNA from CSF depicts a powerful analyte with the potential to alter the standard of care. We have established a robust liquid biopsy program across the neuro-oncology department at MSK, and MSK-IMPACT is now certified by NYS DOH for use on CSF ctDNA enabling routine integration into clinical care. In summary, we are now able to monitor the changing genome along the disease course and have the potential to detect disease occurrence at an earlier time point, however further validation of CSF ctDNA for disease monitoring is needed. Additionally, our data suggests that CSF ctDNA may be used as a prognostic biomarker for survival, but confirmation requires further validation in a prospective study. Citation Format: Subhiksha Nandakumar, Christoph Kreitzer, Charli Ann Hertz, Johnathan Rafailov, Timothy Song, Nicholas D. Socci, A. Rose Brannon, Maria E. Arcila, David B. Solit, Michael F. Berger, Nikolaus Schultz, Ingo K. Mellinghoff, Alexandra M. Miller. Circulating tumor DNA from cerebrospinal fluid (CSF) allows for characterization and monitoring of glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1053.

Efficacy of different platforms in detecting EGFR mutations using cerebrospinal fluid cell-free DNA from non-small-cell lung cancer patients with leptomeningeal metastases.

Cell-free tumor DNA (ctDNA) obtained through liquid biopsy is useful for the molecular analysis of advanced non-small-cell lung cancer (NSCLC). Few studies have directly compared analysis platforms in terms of their diagnostic performance in analyzing ctDNA obtained from the cerebrospinal fluid (CSF) of patients with leptomeningeal metastasis (LM). We prospectively analyzed patients with epidermal growth factor receptor (EGFR)-mutant NSCLC who were subjected to CSF analysis for suspected LM. To detect EGFR mutations, CSF ctDNA was analyzed using the cobas EGFR Mutation Test and droplet digital polymerase chain reaction (ddPCR). CSF samples from osimertinib-refractory patients with LM were also subjected to next-generation sequencing (NGS). Significantly higher rates of valid results (95.1% vs. 78%, respectively, p = 0.04) and EGFR common mutation detection (94.3% vs. 77.1%, respectively, p = 0.047) were obtained through ddPCR than through the cobas EGFR Mutation Test. The sensitivities of ddPCR and cobas were 94.3% and 75.6%, respectively. The concordance rate for EGFR mutation detection through ddPCR and the cobas EGFR Mutation Test was 75.6% and that for EGFR mutation detection in CSF and plasma ctDNA was 28.1%. In osimertinib-resistant CSF samples, all original EGFR mutations were detected through NGS. MET amplification and CCDC6-RET fusion were demonstrated in one patient each (9.1%). The cobas EGFR Mutation Test, ddPCR, and NGS appear to be feasible methods for analyzing CSF ctDNA in patients with NSCLC and LM. In addition, NGS may provide comprehensive information regarding the mechanisms underlying osimertinib resistance.

The diagnostic significance of cerebrospinal fluid cytology and circulating tumor DNA in meningeal carcinomatosis.

The objective of this research is to investigate the clinical application value of cerebrospinal fluid (CSF) cytology and circulating tumor DNA (ctDNA) in lung adenocarcinoma (LUAD) meningeal metastasis-meningeal carcinomatosis (MC), and to further explore the possible molecular mechanisms and drug treatment targets of LUAD meningeal metastasis by next-generation sequencing (NGS). We retrospectively analyzed LUAD with MC in 52 patients. CSF cytology was carried out using the slide centrifugation precipitation method and May-Grüwald-Giemsa (MGG) staining. Tumor tissue, plasma and CSF ctDNA of some MC patients were detected by NGS. Of the 52 MC patients, 46 (88.46%) were positive for CSF cytology and 34 (65.38%) were positive for imaging, with statistically significant differences in diagnostic positivity (P < 0.05). In 32 of these patients, CSF cytology, cerebrospinal fluid ctDNA, plasma ctDNA and MRI examination were performed simultaneously, and the positive rates were 84.38, 100, 56.25, and 62.50% respectively, the difference was statistically significant (P < 0.001). Analysis of the NGS profiles of tumor tissues, plasma and CSF of 12 MC patients: the mutated gene with the highest detection rate was epidermal growth factor receptor (EGFR) and the detection rate were 100, 58.33, and 100% respectively in tumor tissues, plasma and CSF, and there were 6 cases of concordance between plasma and tissue EGFR mutation sites, with a concordance rate of 50.00%, and 12 cases of concordance between CSF and tissue EGFR mutation sites, with a concordance rate of 100%. In addition, mutations not found in tissue or plasma were detected in CSF: FH mutation, SETD2 mutation, WT1 mutation, CDKN2A mutation, CDKN2B mutation, and multiple copy number variants (CNV), with the most detected being CDKN2A mutation and MET amplification. CSF cytology is more sensitive than traditional imaging in the diagnosis of meningeal carcinomatosis and has significant advantages in the early screening and diagnosis of MC patients. CSF ctDNA can be used as a complementary diagnostic method to negative results of CSF cytology and MRI, and CSF ctDNA can be used as an important method for liquid biopsy of patients with MC, which has important clinical significance in revealing the possible molecular mechanisms and drug treatment targets of meningeal metastasis of LUAD.

Early diagnosis of brain metastases using cerebrospinal fluid cell‐free DNA‐based breakpoint motif and mutational features in lung cancer

Early diagnosis of brain metastases using cerebrospinal fluid cell‐free DNA‐based breakpoint motif and mutational features in lung cancer

Abstract P5-05-06: ctDNA detection in seven different types of body liquids in patients with metastatic breast cancer

Abstract Background. Liquid biopsies represent a less invasive alternative to tissue biopsy to characterize and possibly monitor the disease in patients with metastatic breast cancer. So far, blood remains the most frequently investigated body liquid in this context and the investigations mainly focus on the detection, quantification and characterization of the circulating tumor DNA (ctDNA). However, since blood might not capture the full disease profile, other sources of body liquids may have the potential to complement the information obtained from blood. The aims of the present study are therefore to assess whether: (i) ctDNA can be detected in different types of body liquids, and, (ii) the levels of ctDNA in a given liquid are associated with metastases in specific organs. Patients and methods. Twelve patients from the post-mortem tissue donation program UPTIDER (NCT04531696) were included in this study. The receptor status of their primary tumor was: estrogen receptor negative, HER2 non-amplified (ER+/HER2-) (n=9), ER-/HER2- (n=2) and ER+/HER2+ (n=1). Median time between inclusion and death of the patient was 1.6 months (Interquartile range: [0.4-3.4]). Seven types of liquids were collected: blood, saliva, ascites, pleural fluid (PFL), cerebrospinal fluid (CSF), pericardial fluid and urine. Fluids were collected at study inclusion (blood, as well as saliva, urine, and ascites whenever possible) and at autopsy (except for saliva). In total, 108 liquid samples were collected and immediately centrifuged according to standard protocols. Cell free DNA (cfDNA) was extracted from the supernatant. All extracted cfDNA as well as germline DNA extracted from the 12 matched buffy coat samples underwent shallow whole genome sequencing. Log2 ratios were computed with CNVkit, and co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE. Associations between organ involvement and ctDNA yield were assessed by Wilcoxon rank-sum tests. Samples at study inclusion and at autopsy were considered together unless otherwise specified. Results. At the sample level, ctDNA could be identified in 54% of the samples. At the patient level, the proportion of liquid types in which ctDNA was detected was highly variable (median: 58%, IQR: 34-77%, Table 1). CtDNA was detected in ascites of all patients when investigated, in 78% of PFL, 73% of CSF, 67% of blood and 37% of pericardial fluid. Only for one patient with invasive lobular carcinoma, ctDNA was detected in saliva and urine, the latter most likely explained by invasion of the bladder. Of note, in 4/12 patients ctDNA could not be identified in blood but was detected in at least one of the other fluids for 3 of these patients. At autopsy, ctDNA levels tended to be higher in PFL, ascites, and CSF in case of pleural, peritoneal, and central nervous system (CNS) metastases respectively, reaching statistical significance only for PFL. In CSF, two patients have CSF ctDNA detected with no documented involvement of the CNS. No brain autopsy was however performed for these patients. Conclusion. We have shown that ctDNA can be detected in all 7 different body liquids that were investigated in this study. The ctDNA levels in a given liquid can be associated with the presence of metastases in specific organs. Since ctDNA was not detected in 4 of our patients in blood but detectable for 3 of them in other liquids, the evaluation of additional sources of body fluids should be further investigated in patients with metastatic breast cancer. These results therefore open new avenues for the clinical monitoring and characterization of the disease. Table 1. Summary of ctDNA detection per liquid type at the patient level based on the 108 evaluated samples. Histo.= Histological, ILC= Invasive lobular carcinoma, NA= not available, nr= number, NST= non-special type Citation Format: François Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Hajipirloo, Emily Vanden Berghe, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. ctDNA detection in seven different types of body liquids in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-06.

Leptomeningeal disease in histone-mutant gliomas.

The 2016 WHO classification described a subtype of midline gliomas harboring histone 3 (H3) K27M alterations, and the 2021 edition added a new subtype of hemispheric diffuse gliomas with H3 G34R/V mutations. The incidence and clinical behavior of leptomeningeal disease (LMD) in these patients is not well defined. Retrospective study of patients with H3-altered gliomas diagnosed from 01/2012 to 08/2021; histone mutations were identified through next-generation sequencing (NGS) of tumor biopsy and/or cerebrospinal fluid (CSF). We identified 42 patients harboring H3 mutations (K27M mutations in 33 patients, G34R/V in 8, and both in one). Median age was 21 (4-70); 27 were male. LMD was diagnosed in 21/42 (50%) patients, corresponding to a 3-year cumulative incidence of 44.7% (95% confidence interval (CI): 26.1%-63.4%) for the K27-mutant group and a 1-year cumulative incidence of 37.5% in the G34-mutant group (95% CI: 0.01%-74.4%; no events after 1 year). Median time from tumor diagnosis to LMD was 12.9 months for H3-K27 patients and 5.6 months for H3-G34 patients. H3 mutation was detected in CSF in all patients with LMD who had NGS (8 H3-K27-mutant patients). In the H3-K27-mutant group, modeled risk of death was increased in patients who developed LMD (hazard ratio: 7.37, 95% CI: 2.98-18.23, P < .0001). In our cohort, 50% of patients developed LMD. Although further studies are needed, CSF ctDNA characterization may aid in identifying molecular tumor profiles in glioma patients with LMD, and neuroaxis imaging and CSF NGS should be considered for early LMD detection.

Cerebrospinal fluid circulating tumor DNA depicts profiling of brain metastasis in NSCLC.

Brain metastasis (BM) genetically diverges from the primary tumor in non-small-cell lung cancer (NSCLC). Hence, accurately capturing clinically relevant alterations is pivotal for the delivery of targeted therapies. Circulating tumor DNA (ctDNA) sequencing has emerged as a promising liquid biopsy in the biomarker-based clinical management of recurrent and extracranial metastatic NSCLC. However, the absence of simultaneous sequencing data from brain metastatic sites prevents the definitive evaluation of the efficacy of ctDNA in representing genetic profiles in BM. Here, we performed parallel genomic comparisons between matched BM and primary tumor DNA, plasma ctDNA, and cerebrospinal fluid (CSF) ctDNA. The results indicated that CSF ctDNA had a greater ability than plasma ctDNA to comprehensively represent the mutational landscape of BM, with CSF ctDNA detecting all BM mutations in 83.33% of patients, while plasma ctDNA was only 27.78%. Mutant allele frequency (MAF) in CSF ctDNA was highly correlated with the tumor size of BM (r= 0.95), and the mean MAF in CSF ctDNA was higher than that in plasma ctDNA (38.05% vs. 4.57%, respectively). MAF and tumor mutational burden in CSF ctDNA were strongly associated with those in BM (r= 0.96 and 0.97, respectively). Of note, CSF ctDNA had significantly higher concordance with BM than plasma ctDNA (99.33% vs. 67.44%), facilitating the identification of clinically relevant mutations. Moreover, we found that plasma ctDNA has stronger profiling performance, with a concordance of 93.01% in multiple brain metastases, equivalent to CSF ctDNA. Collectively, our study indicates that CSF ctDNA is superior to plasma ctDNA in accurately representing the profiling of single BM. Plasma ctDNA could be an alternative liquid biopsy material to be applied in multiple brain metastatic NSCLC.

Current Role and Future Potential of CSF ctDNA for the Diagnosis and Clinical Management of Pediatric Central Nervous System Tumors.

Most pediatric central nervous system (CNS) tumors are located in eloquent anatomic areas, making surgical resection and, in some cases, even biopsy risky or impossible. This diagnostic predicament coupled with the move toward molecular classification for diagnosis has exposed an urgent need to develop a minimally invasive means to obtain diagnostic information. In non-CNS solid tumors, the detection of circulating tumor DNA (ctDNA) in plasma and other bodily fluids has been incorporated into routine practice and clinical trial design for selection of molecular targeted therapy and longitudinal monitoring. For primary CNS tumors, however, detection of ctDNA in plasma has been challenging. This is likely related at least in part to anatomic factors such as the blood-brain barrier. Due to the proximity of primary CNS tumors to the cerebrospinal fluid (CSF) space, our group and others have turned to CSF as a rich alternative source of ctDNA. Although multiple studies at this time have demonstrated the feasibility of CSF ctDNA detection across multiple types of pediatric CNS tumors, the optimal role and utility of CSF ctDNA in the clinical setting has not been established. This review discusses the work-to-date on CSF ctDNA liquid biopsy in pediatric CNS tumors and the associated technical challenges, and reviews the promising opportunities that lie ahead for integration of CSF ctDNA liquid biopsy into clinical care and clinical trial design.

Dynamic Mutational Landscape of Cerebrospinal Fluid Circulating Tumor DNA and Predictors of Survival after Proton Craniospinal Irradiation for Leptomeningeal Metastases.

Proton craniospinal irradiation (pCSI) is a promising treatment for patients with solid tumor leptomeningeal metastasis (LM). We hypothesize that genetic characteristics before and changes resulting after pCSI will reflect clinical response to pCSI. We analyzed the cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) from patients receiving pCSI for LM and explored genetic variations associated with response. We subjected CSF from 14 patients with LM before and after pCSI to cell-free DNA sequencing using a targeted-sequencing panel. In parallel, plasma ctDNA and primary tumors were subjected to targeted sequencing. Variant allele frequency (VAF) and cancer cell fraction (CCF) were calculated; clonality of observed mutations was determined. Kaplan-Meier analysis was used to associate genomic changes with survival. The median overall survival (OS) for the cohort was 9 months [interquartile range (IQR), 5-21 months]. We showed clonal evolution between tumor and ctDNA of the CSF and plasma with unique mutations identified by compartment. Higher CSF ctDNA mean VAF before pCSI (VAFpre) had worse OS (6 months for VAFpre ≥ 0.32 vs. 9 months for VAFpre < 0.32; P = 0.05). Similarly, increased VAF after pCSI portended worse survival (6 vs. 18 months; P = 0.008). Higher mean CCF of subclonal mutations appearing after pCSI was associated with worse OS (8 vs. 17 months; P = 0.05). In patients with solid tumor LM undergoing pCSI, we found unique genomic profiles associated with pCSI through CSF ctDNA analyses. Patients with reduced genomic diversity within the leptomeningeal compartment demonstrated improved OS after pCSI suggesting that CSF ctDNA analysis may have use in predicting pCSI response.

Noninvasive, Dynamic Risk Profiling of Primary Central Nervous System Lymphoma By Peripheral Blood Ctdna-Sequencing

Introduction: Primary central nervous system lymphoma (PCNSL) is a rare subtype of aggressive B-cell lymphoma restricted to the CNS. Despite the use of intensive chemotherapeutic regimens, relapsed or refractory disease (r/r) occurs frequently and depicts a major challenge. Response assessment is usually based on magnetic resonance imaging (MRI), but highly limited due to insufficient discrimination between scar and active tumor tissue. Furthermore, serial biopsies are often not feasible. Accordingly, there is a high unmet medical need for noninvasive risk profiling strategies in PCNSL. Methods: We applied circulating tumor DNA (ctDNA) sequencing to n=101 blood samples of n=47 patients. Peripheral blood was collected before (pre, n=47), during (mid, n=27) and after (post, n=22) treatment. Sequencing of ctDNA in cerebrospinal fluid (CSF) was performed in 5 patients. Our ctDNA sequencing approach encompasses a targeted gene panel covering >350 genes or genetic regions and an in-house developed bioinformatic pipeline. In the current version, our pipeline shows improved sensitivity and specificity over our previously published version (Sobesky S et al. Med. 2021;2(10):1171-1193.e11), mainly due to the addition of duplex barcode information. Results: The total amount of cell-free DNA (cfDNA) in plasma was generally low (mean 22.3, range 1.4-208.5 ng/ml) as compared to patients with systemic disease. We hypothesized that cfDNA fragment length profiles and their dynamic changes can inform on outcome in PCNSL as has been recently shown for other lymphomas (Meriranta L et al. Blood. 2022;139(12):1863-1877). To this end, we extracted fragment lengths distributions from all cfDNA samples and used principal component analysis to discern fragment length profiles representative of certain types of samples and remission status. Pre-treatment samples contained higher proportions of short cfDNA fragments (133-173 base pairs (bp)) compared to samples obtained mid-treatment, post-treatment, and healthy controls. In contrast, CSF samples were enriched with ultra-short cfDNA fragments (61-97 bp), which were almost absent in all other samples. The detection of single nucleotide polymorphisms (SNP) and small insertions or deletions (InDel) was challenging compared to our previously published experience with Hodgkin lymphoma (Sobesky S et al. Med. 2021;2(10):1171-1193.e11). Nevertheless, we detected mutations in most samples, including recurring mutations in PCNSL-associated genes such as MYD88, PIM1 and CD79, though at a lower frequency than expected. The mean baseline ctDNA concentration was 1.7 (range 0-2.8) log haploid genome equivalents (hGE)/ml plasma. Interestingly, the ctDNA concentration was markedly increased in CSF samples. Higher average mutated allele frequency (mAF) in a plasma sample at baseline was associated with reduced progression-free survival (PFS) and overall survival (OS). Patients with a mAF > 1% showed impaired PFS and OS compared to < 1%. No events were observed in patients with mAF < 0.5% (17% of all patients). MRI-based response evaluation indicated partial response in most patients both mid- and post-treatment and thus failed to predict outcomes. Therefore, we assessed mutation-based tracking of peripheral residual disease (PRD). Persistence of PRD during treatment was associated with impaired PFS (median 3.7 vs. 26.9 months, p < 0.0001) and OS (median 4.7 months vs. not reached, p = 0.0009). We hypothesized that dynamic detection of PRD might complement baseline risk stratification by mAF alone. In our final model, baseline low risk patients (mAF low) became high risk when PRD was positive, and baseline high risk patients (mAF high) became low risk when PRD turned negative during treatment. Strikingly, this classification was highly predictive of both PFS (median 9.3 vs. 26.9 months, p = 0.0004, Fig. 1) and OS (median 10.3 months vs. not reached, p = 0.0010). Conclusions: Surprisingly large differences in fragment length profiles between ctDNA in blood and CSF might yield insight into ctDNA characteristics of both compartments in the context of PCNSL and ctDNA biology in the privileged central nervous system compartment in general. Despite challenges in consistently detecting pathognomonic genetic alterations, assessment of ctDNA-based PRD and fragmentation patterns has the potential to significantly improve treatment guidance in PCNSL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Prospective Study of Methotrexate in Combination with Ibrutinib and Temozolomide (MIT) in Newly Diagnosed Primary CNS Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) is a rare non-Hodgkin lymphoma ,High-dose methotrexate (HD-MTX) systemic chemotherapy is considered as the first-line standard therapy for PCNSL, but the ORR of HD-MTX-based chemotherapy was 60%~70%.The activation of the B-cell receptor pathway has been identified as a key mechanism of PCNSL and BTK inhibitor is a promising agent. Here we aimed to investigate the safety and efficacy of induction treatment, high-dose methotrexate in combination with ibrutinib and temozolomide (MIT) in newly diagnosed PCNSL. Methods: This open-label， single-arm, multi-center phase II prospective study(NCT04514393) was designed to enroll 33 patients , and 25 patients have response (approx 75%), the MIT treatment would be considered effective. Eligible patients were ≥18 and ≤75 years of age with adequate organ function, with at least one measurable lesion. The MIT induction treatment included six cycles of high-dose methotrexate (HD-MTX, 3.5 g/m2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients < 65 yr) and daily ibrutinib (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of the induction MIT treatment. The primary endpoint was the overall response rate (ORR) of the 6 cycles of MIT induction treatment for PCNSL evaluated by investigator. The safety of the treatment were also investigated. Baseline and sequential CSF/plasma samples were collected and underwent targeted next-generation sequencing (NGS). Results: Between July 2021 and July 2022, thirty-one newly diagnosed PCNSL patients with no prior therapy were enrolled and 28 patients accepted at least 2 cycles of MIT treatment. The median age of the patients was 53 yr (range: 27-75) and15/28 (53.6%) were male. By data cutoff, 12/28 (42.9%) patients had completed six cycles of treatment, 9/28 had completed 4 cycles of treatment, and 7/28 had completed 2 cycles of treatment. The ORR was 92.9% (26/28), and CR rate was 67.9% (19/28). Among 12 patients had completed 6 cycles of treatment, 11/12 (91.7%) patients achieved CR, 1 patient achived PR. Among 9 patients had completed 4 cycles of treatment, 4 (44.4%) achived CR, 3/9 (33.3%) achived PR. Among 7 patients had completed 2 cycles of treatment, 4 (57.1%) patients achieved CR and 3 (42.8%) pateints achived PR. The mutation profiles of dynamic CFS and plasma ctDNA were collected and analyzed. The most commonly mutated genes in CSF were PIM1, MYD88, BTG2, CD79B, IRF4, and ETS1. The Cerebrospinal fluid protein (CSF-TP) and CSF cfDNA concentration (ng/ml), ctDNA MAF, and ctDNA concentration (hGe/ml), MAF,and hGe/ml were positively correlated. We compared gene profiles of patients in response and no response. KMT2D gene mutations were more commonly detected in patients with progression disease compared to patients responding to treatment (P=0.03). Interestingly, detectable baseline plasma ctDNA and post-treatment CSF ctDNA were significant associated with progression disease (P=0.03, P=0.004, respectively). This clinical trial is still on going. Conclusions: The MIT induction treatment could achieve higher responses compared with HD-MTX-based chemotherapy in newly diagnosed PCNSL patients and the adverse events are manageable.

Preliminary Results of Penpulimab Combined with RMA (rituximab, methotrexate, and cytarabine) for Newly Diagnosed Primary Central Nervous System Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) has been classified as large B-cell lymphomas (LBCL) of immune-privileged sites according to the 5th edition of the World Health Organization (WHO) Classification. The efficacy of high-dose methotrexate as a first-line treatment was suboptimal with a short effective remission time and low response rate. The recent discovery of the frequent copy number alterations in 9p24.1/PD-L1/PD-L2 and increased expression of PD-L1 in PCNSL provide the rationale to evaluate the efficacy of PD-1antibody in PCNSL. Methods: NCT05347641 is an ongoing Phase II, single-center, single-arm, open-label study, aimed to enrolled an estimated 23 newly diagnosed PCNSL patients. Enrolled patients first received the Pen-RMA induction treatment, including rituximab 375 mg/m2, d0; methotrexate 3.5g/m2, d1; cytarabine 1‒2g/m2, q12h, d2-3; penpulimab (an anti-PD1 antibody of IgG1 isotype) 200 mg, d5; 21d/cycle, for six cycles. Patients aged 60 years or younger, with complete response (CR)/unconfirmed complete response (CRu)/partial response (PR) after six cycles of induction treatment then underwent autologous stem cell transplantation (ASCT) before maintenance treatment. The maintenance regimen was administered based on patient response to ASCT, or to induction therapy for patients aged 60 years or older. Patients who achieved CR after ASCT or induction therapy then received 8 cycles of penpulimab as maintenance therapy, and those with PR after ASCT or induction therapy received whole brain radiotherapy (WBRT) combined with 8 cycles of penpulimab as maintenance. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every three cycles of the induction treatment. The primary endpoint of this study was two-year progression-free survival rate. Baseline tissue samples and sequential CSF or plasma samples were collected for targeted next-generation sequencing (NGS) at Nanjing Geneseeq Technology Inc. Results: Between April, 2022 and July, 2022, eleven newly diagnosed PCNSL patients has been enrolled. Treatment response was evaluable in eight patients. At the interim assessment point (after 3 cycles), six patients have achieved CR, with an overall response rate of 75.0%. Of these, three have completed 6 cycles of treatment and all achieved CR at the final assessment (Figure A). Adverse events of grade 3 or 4 occurred in five patients, including three neutropenia (27.3%), one thrombocytopenia (9.1%), and one renal dysfunction (9.1%). In the baseline samples, PIM1, MYD88L265P, and CD79B were the most common mutations, with a more comprehensive genomic landscape revealed by CSF than by blood (Figure B). All nine NGS-tested patients were classifier into the non-GCB subtype by the COO classification and the MCD subtype by the LymphGen classifier. CSF-based ctDNA monitoring was performed in two patients after 6 cycles of treatment. P01 had confirmed CR with ctDNA clearance in CSF. P07, was assessed as CR after 6 cycles, but the CSF ctDNA remained positive. This patient is currently receiving maintenance treatment and is being closely monitored for potential disease relapse. Interestingly, P02 and P04, who were PD and SD at interim evaluation, respectively, had markedly more mutations detected in their baseline plasma samples, indicating a poor prognosis (Figure A). Conclusion: The induction treatment of Pen-RMA has shown excellent responses in newly diagnosed PCNSL patients with mild hematologic toxicity. Our preliminary results also suggested that CSF-based ctDNA could facilitate diagnosis when tissue was inaccessible and show higher sensitivity than imageology in assessing the depth of remission. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamic monitoring of cerebrospinal fluid circulating tumor DNA to identify unique genetic profiles of brain metastatic tumors and better predict intracranial tumor responses in non-small cell lung cancer patients with brain metastases: a prospective cohort study (GASTO 1028)

BackgroundDue to the blood-brain barrier, plasma is not an ideal source to evaluate the genetic characteristics of central nervous system tumors. Thus, cerebrospinal fluid (CSF) is becoming an alternative biopsy type to evaluate the genetic landscape of intracranial tumors. We aimed to explore the genetic profiles of CSF-derived circulating tumor DNA (ctDNA) to predict intracranial tumor responses and monitor mutational evolution during the treatment of non-small cell lung cancer (NSCLC) patients with brain metastases.MethodsWe conducted a prospective study of 92 newly diagnosed NSCLC patients with brain metastases. Paired CSF and plasma samples were collected at baseline, 8 weeks after treatment initiation, and disease progression. All samples underwent next-generation sequencing of 425 cancer-related genes.ResultsAt baseline, the positive detection rates of ctDNA in CSF, plasma, and extracranial tumors were 63.7% (58/91), 91.1% (82/90), and 100% (58/58), respectively. A high level of genetic heterogeneity was observed between paired CSF and plasma, while concordance in driver mutations was also observed. A higher number of unique copy number variations was detected in CSF-ctDNA than in plasma. ctDNA positivity of CSF samples at baseline was associated with poor outcomes (HR=2.565, P=0.003). Moreover, patients with ≥ 50% reductions in the concentrations of CSF ctDNA after 8 weeks of treatment had significantly longer intracranial progression-free survivals (PFS) than patients with < 50% reductions in CSF ctDNA concentrations (13.27 months vs 6.13 months, HR=0.308, P=0.017). A ≥ 50% reduction in CSF ctDNA concentrations had better concordance with radiographic intracranial tumor responses than plasma. A ≥ 50% reduction in plasma ctDNA concentrations was also associated with longer extracranial PFS (11.57 months vs 6.20 months, HR=0.406, P=0.033). Based on clonal evolution analyses, the accumulation of subclonal mutations in CSF ctDNA was observed after 8 weeks of treatment. The clonal mutations that remained in more than 80% in CSF after 8 weeks also predicted shorter intracranial PFS (HR=3.785, P=0.039).ConclusionsCSF ctDNA exhibited unique genetic profiles of brain metastases, and dynamic changes in CSF ctDNA could better predict intracranial tumor responses and track clonal evolution during treatment in NSCLC patients with brain metastases.Trial registrationClinicalTrials.gov identifier: NCT 03257735.

CNSC-04. CEREBROSPINAL FLUID-DERIVED CIRCULATING TUMOR DNA IS SUPERIOR TO PLASMA IN NSCLC PATIENTS WITH CNS PROGRESSION

Abstract Introduction Central nervous system (CNS) metastases are devastating neurologic complications. However, the clinical value of cerebrospinal fluid (CSF) as liquid biopsy medium in EGFR-mutated or ALK-rearranged patients with brain metastases (BM) or leptomeningeal metastases (LM) remains unclear. Patients and Methods We retrospectively analyzed the gene map of 14/48 NSCLC-BM patients and 34/48 NSCLC-LM patients. Compare the clinical predictive value of CSF and matched plasma (PLA) for patients with single CNS progression (cohort 1) or intracranial progression simultaneously with extracranial disease progression (cohort 2). Circulating cell-free tumor DNA (ctDNA) in CSF and matched PLA underwent next-generation sequencing targeting 168 cancer-relevant genes. Results For patients with single CNS progression, the mutation rate of major driver genes is much higher than the rate in matched PLA. Besides, CSF also demonstrated higher levels of potential prognostic markers in comparison with matched PLA samples, showing EGFR-amp presented more than two times the rate compared to matched PLA, and the detection rate of TBX3, PTEN were almost double the rate in CSF than paired PLA samples. In addition, poor outcomes associated with makers including CDKN2A/B, PIK3CA/G, CDK4/6, and MET showed exclusively in CSF samples.Almost all mutations identified in the PLA could detect in matched CSF samples in NSCLC-LM patients with both intracranial and extracranial disease progression. In addition, detection rates of driver mutations, consisting of EGFR or ALK are much higher in CSF compared to matched PLA, and these potential survival marker genes like CDK4/6, CDKN2A/B, EGFR-amp, MET, and PIK3CA identified uniquely in CSF samples. Conclusion CSF is more comprehensive in genomic mutation profile than matched PLA in NSCLC -LM regardless of single intracranial progression or intracranial progression simultaneously with extracranial evolution.

Tapping into the genome: the role of CSF ctDNA liquid biopsy in glioma.

Liquid biopsy has emerged as a novel noninvasive tool in cancer diagnostics. While significant strides have been made in other malignancies using liquid biopsy for diagnosis, disease monitoring, and treatment selection, development of these assays has been more challenging for brain tumors. Recently, research in primary and metastatic brain tumors has begun to harness the potential utility of liquid biopsy-particularly using circulating tumor DNA (ctDNA). Initial studies to identify ctDNA in plasma of brain tumor patients have shown feasibility, but the yield of ctDNA is far below that for other malignancies. Attention has therefore turned to the cerebrospinal fluid (CSF) as a more robust source of ctDNA. This review discusses the unique considerations in liquid biopsy for glioma and places them in the context of the work to date. We address the utility of CSF liquid biopsy for diagnosis, longitudinal monitoring, tracking tumor evolution, clinical trial eligibility, and prognostication. We discuss the differences in assay requirements for each clinical application to best optimize factors such as efficacy, cost, and speed. Ultimately, CSF liquid biopsy has the potential to transform how we manage primary brain tumor patients.