Cerebrospinal Fluid Circulating Tumor DNA Research Articles

Combination of Cytologic Findings and Circulating Tumor DNA From Cerebrospinal Fluid Revealed SCLC Transformation in Patients With Leptomeningeal Metastases of Lung Adenocarcinoma

IntroductionTransformation to SCLC is a resistance mechanism to tyrosine kinase inhibitor in EGFR-mutated lung adenocarcinoma (LUAD). Nevertheless, the clinical and molecular features of SCLC transformation in LUAD with leptomeningeal metastases (LM) are scarce. MethodsWe retrospectively collected 237 patients with NSCLC who underwent lumbar puncture owing to suggestion of LM. All SCLC transformation in cerebrospinal fluid (CSF) was confirmed by two experienced pathologists using cytologic evaluation. CSF circulating tumor DNA (ctDNA) was tested by next-generation sequencing. ResultsTumor cells in CSF samples were found in 111 patients (111 of 237, 46.8%), and eight cases (eight of 111, 7.2%) were identified as having SCLC cells in CSF. Seven patients carried the EGFR mutation, including four patients with EGFR exon 19 deletion and three patients with EGFRexon21 L858R mutation. Another patient harbored ERBB2 insertion. Seven of these patients were resistant to targeted therapy. CSF ctDNA analysis reported that TP53 and RB1 mutations were common. The median time from the diagnosis of advanced NSCLC to SCLC transformation found in CSF was 9.7 months (95% confidence interval [CI]: 4.0–17.5 mo). The median overall survival since the initial diagnosis of metastatic NSCLC was 15.3 months (95% CI: 1.2–29.4 mo). The median overall survival after SCLC transformation detected in CSF was 5.0 months (95% CI: 4.0–5.9 mo). ConclusionsSCLC transformation may be revealed in CSF by both cytologic evaluation and ctDNA, not just in tissue that underwent rebiopsy. SCLC transformation of CSF is informative for resistance mechanism in patients with LUAD with LM on tyrosine kinase inhibitor progression, which was associated with poor survival.

Abstract 2421: Targeted and low-pass whole genome sequencing of cerebrospinal fluid circulating tumor DNA for the diagnosis and monitoring of pediatric, adolescent and young adult CNS tumors

Abstract Molecular analysis of CNS tumors is essential to their diagnosis and guides individualized treatment for patients, but tissue biopsy of primary and/or relapsed lesions to allow for this characterization often carries intolerable risk. The analysis of genetic material in cerebrospinal fluid (CSF), known as liquid biopsy, can allow for minimally invasive tumor identification and diagnosis. Furthermore, liquid biopsy can serve as a more sensitive test for residual disease than imaging or cytologic analyses. We aimed to implement CSF liquid biopsy at Canada’s largest pediatric hospital. >150 CSF samples were collected from CNS tumor patients with a median age of 9. The most common tumor types were low-grade gliomas (40%), high-grade gliomas (21%) and medulloblastomas (MB) (17%). The majority were collected via lumbar puncture (46%), external ventricular drain (24%) and intraoperatively (22%). The most frequent collection times were diagnosis (53%) and progression/relapse (28%). Cell-free DNA was extracted, and circulating tumor DNA was analyzed using low-pass whole genome sequencing and a targeted hybridization capture glioma gene panel for select cases. Pathogenic mutations and copy number alterations (CNA) were called using an in-house bioinformatics pipeline. Somatic alterations were detected in 57% of samples from patients with pathologically confirmed CNS tumors. CNAs were identified in 5/7 MB patients at the time of pre-treatment staging lumbar puncture. We also detected CNAs in several MB patients during treatment and at progression/relapse, including a patient with disseminated MB who was mid-induction chemotherapy with 1 suspicious cell in their CSF, highlighting our ability to detect minimal residual disease. Among samples with no previous biopsy, we identified tumor DNA in 29%, often providing diagnostic clarity and guiding management. For example, we identified a targetable FGFR1 N546D missense mutation in a patient with an unbiopsied suprasellar lesion. We also diagnosed recurrent MB in a patient 6 years off treatment with a new ambiguous nodule in their tumor bed by detecting an isochromosome 17q CNA. Overall, our liquid biopsy platform is feasible and can yield clinically impactful results. Next steps include refining our assays to increase their sensitivity and systematically evaluating our platform’s efficacy for monitoring response to treatment and tumor surveillance. Citation Format: Ian Burns, Liana Nobre, Yoshiko Nakano, Michelle Ku, Monique Johnson, Javal Sheth, Logine Negm, Chantel Cacciotti, Mansuba Rana, Richard Yuditskiy, Andrew Bondoc, Robert Siddaway, Uri Tabori, Cynthia Hawkins. Targeted and low-pass whole genome sequencing of cerebrospinal fluid circulating tumor DNA for the diagnosis and monitoring of pediatric, adolescent and young adult CNS tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2421.

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

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. Patients with LM from NSCLC (n = 80) were retrospectively analyzed. Circulating tumor DNA (ctDNA) in CSF was tested by next-generation sequencing (NGS), with paired extracranial tissue or plasma samples included for comparison. An independent non-LM cohort (n = 100) was also analyzed for comparative purposes. Clinical outcomes were compared with Kaplan-Meier log-rank test and Cox proportional hazards methodologies. An overwhelming 93.8% of patients carried druggable mutations in NSCLC LM, with EGFR (78.8%) being the most prevalent. Notably, 4 patients who tested negative for driver genes in extracranial samples surprisingly showed EGFR mutations in their CSF and subsequently benefited from targeted therapy. There was a clear difference in genetic profiles between CSF and extracranial samples, with CSF showing more driver gene detections, increased Copy Number Variations (CNVs), and varied resistance mechanisms among individuals. Abnormalities in cell-cycle regulatory molecules were highly enriched in LM (50.9% vs 31.0%, p = 0.017), and CDKN2A/2B deletions were identified as an independent poor prognostic factor for LM patients, with a significant reduction in median OS (p = 0.013), supported by multivariate analysis (HR 2.63, 95% CI 1.32-5.26, p = 0.006). CSF-based ctDNA analysis is crucial for detecting and characterizing genetic alterations in NSCLC LM. The distinct genetic profiles in CSF and extracranial tissues emphasize the need for personalized treatment approaches.

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.

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.

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.

Orelabrutinib Combined With Lenalidomide and Immunochemotherapy for Relapsed/Refractory Primary Central Nervous System Lymphoma: A Retrospective Analysis of Case Series.

BackgroundRelapsed/refractory (r/r) primary central nervous system lymphoma (PCNSL) is an intractable situation without sound treatment. Bruton’s tyrosine kinase (BTK) represents an attractive drug target in PCNSL. Orelabrutinib is a new-generation BTK inhibitor with high cerebrospinal fluid (CSF) concentration. This study aimed to evaluate the efficacy and safety of orelabrutinib-containing combination therapy in patients with r/r PCNSL.MethodsWe retrospectively analyzed r/r PCNSL patients who received combination therapy with rituximab, high-dose methotrexate, temozolomide, orelabrutinib and lenalidomide, and further explored the relationship between the efficacy and genetic characteristics.ResultsA total of fifteen patients were included in this retrospective study. The overall response rate (ORR) was 86.7%, the complete remission (CR) rate was 73.3% and the disease control rate (DCR) was 93.3%. Among 13 responders, 9 patients are still receiving oral orelabrutinib and lenalidomide. The most common adverse event (AEs) was transaminase increase (66.7%). No grade 4 AE or drug-related death was reported. Genomic sequencing showed that patients who responded to orelabrutinib had abnormal NF-κB activation, while those who had no response were mainly enriched with transcriptional misregulation. Patients who had mutations in TLR, BCR, or NF-κB pathway achieved complete or partial response to the orelabrutinib-containing therapy. Moreover, the blood and cerebrospinal fluid circulating tumor DNA (ctDNA) were closely associated with tumor recurrence and treatment response and sustained tumor responses correlated with the clearance of ctDNA.ConclusionOrelabrutinib-containing regimen was effective and well-tolerated in patients with r/r PCNSL. Genome sequencing of tumor samples could help to screen patients who may respond to the orelabrutinib-containing regimen, and liquid biopsy may contribute to tracing tumor burden and monitoring treatment response.

Dynamic monitoring of cerebrospinal fluid circulating tumor DNA to identify unique genetic profiles of brain metastatic tumors and to better predict intracranial tumor response in patients with non–small cell lung cancer with brain metastases: A prospective cohort study.

2023 Background: Cerebrospinal fluid (CSF) can be used as a type of liquid biopsy to detect brain tumors. We aimed to explore the genetic profiles of CSF-derived circulating tumor DNA (ctDNA) to predict intracranial tumor response and monitor mutational evolution during systemic treatment in non-small cell lung cancer (NSCLC) patients with brain metastases. Methods: We conducted aprospective 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 at disease progression. Primary extracranial tumor samples were available for 58 patients and all samples underwent targeted next generation sequencing of 425 cancer-related genes. Results: At 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 heterogeneity was observed between paired CSF and plasma samples, while concordance in driver mutations was also observed. A higher number of unique copy number variations (CNVs) were detected in CSF ctDNA than in plasma. CtDNA-positivity in baseline CSF samples was associated with poor outcomes (HR = 2.565, P = 0.003). Moreover, patients with increased concentrations of ctDNA in CSF after 8 weeks of treatment had significantly shorter intracranial progression-free survivals (PFS) than patients with decreased concentrations of CSF ctDNA (6.13 months vs 13.27 months, HR = 3.92, P = 0.007). Increased concentrations of plasma ctDNA were associated with shorter extracranial PFS (6.13 months vs 11.57 months, HR = 2.626, P = 0.032). From clonal evolution analyses, the accumulation of subclonal mutations in CSF ctDNA was observed after 8 weeks of systemic treatment. The clonal mutations remained more than 80% in CSF after 8-weeks of treatment also predicted a shorter intracranial PFS (HR = 3.785, P = 0.039). Conclusions: CSF ctDNA revealed unique genetic profiles of brain metastases, and dynamic changes in CSF ctDNA could better predict intracranial tumor response and track clonal evolution during treatment in NSCLC patients with brain metastases.

BrainStorm: A brain metastases research platform to tackle the challenge of central nervous system (CNS) metastases in solid tumors (Oncodistinct 006).

TPS2066 Background: Better knowledge on the evolving epidemiology and biology of CNS metastases are key elements for the development of new treatment strategies and identification of promising therapeutic targets. A multidisciplinary Brain Metastases Clinical Research Platform called BrainStorm was launched by the Jules Bordet Institute and the Oncodistinct network. The BrainStorm program includes mainly patients with non-CNS metastatic solid tumors with high risk of developing CNS metastases allowing to build a large database focusing on three time periods: before the diagnosis of CNS metastases (Part A), at diagnosis (Part B) and after the diagnosis of CNS metastases (Part C). Methods: Subjects with newly diagnosed non-CNS metastases or up to 24 months from diagnosis of non-CNS metastases from triple-negative and HER2-positive breast cancer, lung cancer and melanoma are eligible for part A of the program. Subjects presenting with a 1st CNS event and not yet enrolled (previously mentioned cohorts and a cohort of other tumor types) can enter directly in parts B and subsequently part C of the study. Eligible subjects are followed for 48 months for relevant clinical data, neurological examinations, quality of life, survival status, and undergo examinations and samplings (Table 1). A total of 280 subjects (40 per cohort) with a 1st CNS event will be enrolled. The main objectives of the program are to collect clinical and biological data with the aim to identify risk factors for CNS metastases development (Part A) and to better understand the biology of CNS metastases (brain and leptomeningeal –Parts B and C) aiming to discover new targets for therapy and intensify the multidisciplinary approach for the management of CNS metastases from solid tumors. The translational part of the program will evaluate among others the use of cerebrospinal fluid circulating tumor DNA (CSF-ctDNA) as a surrogate for CNS metastases in order to characterize its molecular landscape. Currently, the study is recruiting in several sites within the Oncodistinct network. The data collected will help to develop innovative multidisciplinary research projects that could be implemented in all parts of the BrainStorm program. Clinical trial information: NCT04109131. [Table: see text]

Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next generation sequencing

BackgroundThis study profiled the somatic genes mutations and the copy number variations (CNVs) in cerebrospinal fluid (CSF)-circulating tumor DNA (ctDNA) from patients with neoplastic meningitis (NM).MethodsA total of 62 CSF ctDNA samples were collected from 58 NM patients for the next generation sequencing. The data were bioinformatically analyzed by (Database for Annotation, Visualization and Integrated Discovery) DAVID software.ResultsThe most common mutated gene was TP53 (54/62; 87.10%), followed by EGFR (44/62; 70.97%), PTEN (39/62; 62.90%), CDKN2A (32/62; 51.61%), APC (27/62: 43.55%), TET2 (27/62; 43.55%), GNAQ (18/62; 29.03%), NOTCH1 (17/62; 27.42%), VHL (17/62; 27.42%), FLT3 (16/62; 25.81%), PTCH1 (15/62; 24.19%), BRCA2 (13/62; 20.97%), KDR (10/62; 16.13%), KIT (9/62; 14.52%), MLH1 (9/62; 14.52%), ATM (8/62; 12.90%), CBL (8/62; 12.90%), and DNMT3A (7/62; 11.29%). The mutated genes were enriched in the PI3K-Akt signaling pathway by the KEGG pathway analysis. Furthermore, the CNVs of these genes were also identified in these 62 samples. The mutated genes in CSF samples receiving intrathecal chemotherapy and systemic therapy were enriched in the ERK1/2 signaling pathway.ConclusionsThis study identified genes mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.

Applications of cerebrospinal fluid circulating tumor DNA in the diagnosis of gliomas.

The 2016 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) was revised to include molecular biomarkers as diagnostic criteria. However, conventional biopsies of gliomas were spatially and temporally limited. This study aimed to determine whether circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) could provide more comprehensive diagnostic information to gliomas. Combined with clinical data, we analyzed gene alterations from CSF and tumor tissues of newly diagnosed patients, and detected mutations of ctDNA in recurrent patients. We simultaneously analyzed mutations of ctDNA in different glioma subtypes, and in lower-grade gliomas (LrGG) versus glioblastoma multiforme (GBM). CSF ctDNA mutations had high concordance rates with tumor DNA (tDNA). CSF ctDNA mutations of PTEN and TP53 were commonly detected in recurrent gliomas patients. IDH mutation was detected in most of CSF ctDNA derived from IDH-mutant diffuse astrocytomas, while CSF ctDNA mutations of RB1 and EGFR were found in IDH-wild-type GBM. IDH mutation was detected in LrGG, whereas Rb1 mutation was more commonly detected in GBM. CSF ctDNA detection can be an alternative method as liquid biopsy in gliomas.