ABSTRACT
Leptomeningeal metastasis (LM) is a lethal complication of malignant tumors, with an incidence rate of 3%-5% among patients with non-small cell lung cancer (NSCLC). LM poses significant challenges in diagnosis, has poor prognosis, limited treatment options, and lacks standardized criteria for evaluating therapeutic efficacy, making it a difficult aspect of NSCLC management. Circulating tumor DNA (ctDNA), shed from tumor cells and carrying cancer-related information, holds significant value in precision oncology. Cerebrospinal fluid (CSF), present in the subarachnoid space of the brain, the spinal cord, and the central canal, and in direct contact with meningeal tissues, serves as the fluid medium that best reflects the genetic characteristics of LM. In recent years, CSF ctDNA has become a focal point due to its multi-omics features, playing a crucial role in the management of central nervous system (CNS) metastatic tumors. Its applications span the entire continuum of care, including aiding in diagnosis, assessing treatment response, predicting prognosis, and analyzing resistance mechanisms. This article provides a concise overview of CSF ctDNA detection techniques and their clinical applications in patients with NSCLC-LM.â©.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Circulating Tumor DNA , Lung Neoplasms , Meningeal Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Circulating Tumor DNA/cerebrospinal fluid , Circulating Tumor DNA/genetics , Circulating Tumor DNA/blood , Lung Neoplasms/pathology , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Meningeal Neoplasms/secondary , Meningeal Neoplasms/cerebrospinal fluid , Meningeal Neoplasms/geneticsABSTRACT
Leptomeningeal metastases (LM) occur in patients with breast cancer (BC) and lung cancer (LC) showing exceptionally poor prognosis. The cerebrospinal fluid (CSF) tumour microenvironment (TME) of LM patients is not well defined at a single-cell level. Based on the 10× genomics single-cell RNA sequencing (scRNA-seq) data from GEO database including five patient-derived CSF samples of BC-LM and LC-LM, and four patient-derived CSF samples of idiopathic intracranial hypertension (IIH) as controls, we analysed single-cell transcriptome characteristics of CSF TME in LM patients compared to controls simultaneously and comprehensively. In addition, we performed 10× genomics scRNA-seq on CSF cells derived from a BC-LM patient to help generate a solid conclusion. The CSF macrophages in LM patients showing M2-subtype signature and the emergence of regulatory T cells in LM confirmed the direction of tumour immunity toward immunosuppression. Then, the characteristics of CSF circulating tumour cells (CTCs) of breast cancer LM (BC-LM) patients were classified into five molecular subtypes by PAM50 model. The communication between macrophages and five subtype-specific CSF-CTCs showed largest number of ligand-receptor interactions. The five subtypes-specific CSF-CTCs showed great heterogeneities which were manifested in cell proliferation and cancer-testis antigens expression. Gene regulatory networks (GRNs) analysis revealed that transcription factor SREBF2 was universally activated in the five subtypes-specific CSF-CTCs. Our results will provide inspiration on new directions of the mechanism research, diagnosis and therapy of LM.
Subject(s)
Breast Neoplasms , Lung Neoplasms , Meningeal Carcinomatosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Genomics , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Sequence Analysis, RNA , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: Brain metastasis is a common and lethal complication of non-small cell lung cancer (NSCLC). It is mostly diagnosed only after symptoms develop, at which point very few treatment options are available. Therefore, patients who have an increased risk of developing brain metastasis need to be identified early. Our study aimed to identify genomic and epigenomic biomarkers for predicting brain metastasis risk in NSCLC patients. METHODS: Paired primary lung tumor tissues and either brain metastatic tissues or cerebrospinal fluid (CSF) samples were collected from 29 patients with treatment-naïve advanced NSCLC with central nervous system (CNS) metastases. A control group comprising 31 patients with advanced NSCLC who died without ever developing CNS metastasis was also included. Somatic mutations and DNA methylation levels were examined through capture-based targeted sequencing with a 520-gene panel and targeted bisulfite sequencing with an 80,672 CpG panel. RESULTS: Compared to primary lung lesions, brain metastatic tissues harbored numerous unique copy number variations. The tumor mutational burden was comparable between brain metastatic tissue (P = 0.168)/CSF (P = 0.445) and their paired primary lung tumor samples. Kelch-like ECH-associated protein (KEAP1) mutations were detected in primary lung tumor and brain metastatic tissue samples of patients with brain metastasis. KEAP1 mutation rate was significantly higher in patients with brain metastasis than those without (P = 0.031). DNA methylation analysis revealed 15 differentially methylated blocks between primary lung tumors of patients with and without CNS metastasis. A brain metastasis risk prediction model based on these 15 differentially methylated blocks had an area under the curve of 0.94, with 87.1% sensitivity and 82.8% specificity. CONCLUSIONS: Our analyses revealed 15 differentially methylated blocks in primary lung tumor tissues, which can differentiate patients with and without CNS metastasis. These differentially methylated blocks may serve as predictive biomarkers for the risk of developing CNS metastasis in NSCLC. Additional larger studies are needed to validate the predictive value of these markers.
Subject(s)
Brain Neoplasms , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Brain Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA Copy Number Variations/genetics , DNA Methylation/genetics , Genomics , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mutation/genetics , NF-E2-Related Factor 2/geneticsABSTRACT
Lung adenocarcinoma (LUAD) is most common pathological type of lung cancer. LUAD with brain metastases (BMs) usually have poor prognosis. To identify the potential genetic factors associated with BM, a genomic comparison for BM cerebrospinal fluid (CSF) and primary lung tumor samples obtained from 1082 early- and late-stage LUAD patients was performed. We found that single nucleotide variation (SNV) of EGFR was highly enriched in CSF (87% of samples). Compared with the other primary lung tissues, copy number gain of EGFR (27%), CDK4 (11%), PMS2 (11%), MET (10%), IL7R (8%), RICTOR (7%), FLT4 (5%), and FGFR4 (4%), and copy number loss of CDKN2A (28%) and CDKN2B (18%) were remarkably more frequent in CSF samples. CSF had significantly lower tumor mutation burden (TMB) level but more abundant copy number variant. It was also found that the relationships among co-occurrent and mutually exclusive genes were dynamically changing with LUAD development. Additionally, CSF (97% of samples) harbored more abundant targeted drugs related driver and fusion genes. The signature 15 associated with defective DNA mismatch repair (dMMR) was only identified in the CSF group. Cancer associated pathway analysis further revealed that ErbB (95%) and cell cycle (84%) were unique pathways in CSF samples. The tumor evolution analysis showed that CSF carried significantly fewer clusters, but subclonal proportion of EGFR was remarkably increased with tumor progression. Collectively, CSF sequencing showed unique genomic characteristics and the intense copy number instability associated with cell cycle disorder and dMMR might be the crucial genetic factors in BM of LUAD.
Subject(s)
Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/genetics , Brain Neoplasms/cerebrospinal fluid , Brain Neoplasms/genetics , Genetic Predisposition to Disease , Genomics , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Adolescent , Adult , Aged , Clone Cells , DNA Copy Number Variations/genetics , Female , Genotype , Humans , Liver/metabolism , Liver/pathology , Lung/pathology , Male , Middle Aged , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Signal Transduction/genetics , Tumor Burden/genetics , Young AdultABSTRACT
BACKGROUND: Tumor markers (TM) in cerebrospinal fluid (CSF) are useful for diagnosing leptomeningeal metastasis (LM). It has not been fully exploited the diagnostic possibilities of the CSF levels since the basic fact that the TM concentration of CSF depends strongly upon the serum levels as well as upon the condition of the blood brain barrier (BBB). To analyze the intrathecal TM synthesis and evaluate the integrity of BBB can be helpful for the definitive diagnosis of LM. Therefore, the aim of this study was to further explore the clinical value of intrathecal TM synthesis and BBB in the diagnosis for the lung cancer patients with LM. METHODS: Twenty-five lung cancer patients with LM and 57 patients with nonmalignant neurological diseases (NMNDs) admitted to Nanjing Drum Tower Hospital from December 2016 to March 2020 were included. We compared the integrity of BBB and intrathecal TM synthesis between two groups, analyzed the correlation of CSF TM between the detection and intrathecal synthesis, and evaluated serial CSF cytology, the integrity of BBB and intrathecal TM synthesis when intrathecal chemotherapy for one patient. RESULTS: Ninety-four percent LM patients showed the dysfunction of BBB, and all LM patients showed at least one intrathecal synthesized TM in CSF. In one patient, the CSF cytology was negative for the first time, but LM was eventually diagnosed based on the the intrathecal TM synthesis and positive CSF cytology of repeated lumbar puncture. In LM group, no correlation was observed between the detection and intrathecal synthesized TM in CSF. In the control group, only 3.5% (2/57) NMNDs patients had the dysfunction of BBB and no patients had intrathecal TM synthesis, both the differences of which were statistically significant (P<0.05). Finally, evaluating the CSF cytology, integrity of BBB and intrathecal TM synthesis can be used to assess the intracranial treatment effect. Moreover, intrathecal TM synthesis changes earlier than cytology. CONCLUSIONS: The evaluation of intrathecal TM synthesis and integrity of BBB are novel clinical diagnostic tools. In addition, serial measurement of intrathecal synthesized TM may play an important role in monitoring efficacy of lung cancer patients with LM, which is worthy of further promotion and clinical application.
Subject(s)
Biomarkers, Tumor , Lung Neoplasms , Meningeal Neoplasms , Adult , Aged , Biomarkers, Tumor/blood , Biomarkers, Tumor/cerebrospinal fluid , Blood-Brain Barrier/physiopathology , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/diagnosis , Lung Neoplasms/physiopathology , Male , Meningeal Carcinomatosis/blood , Meningeal Carcinomatosis/cerebrospinal fluid , Meningeal Carcinomatosis/physiopathology , Meningeal Carcinomatosis/secondary , Meningeal Neoplasms/blood , Meningeal Neoplasms/cerebrospinal fluid , Meningeal Neoplasms/physiopathology , Meningeal Neoplasms/secondary , Middle AgedABSTRACT
Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first- and second-generation ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3, and CCND1. Specifically, a deletion in exon 19 in EGFR, a non-V600 BRAF mutation (G466V), and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK-Is. Potential ALK-I-resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and FGFR3, was detected in a patient progressing on a first-line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK-I-resistance mutations in most cases and could be a valuable approach for therapy decision making.
Subject(s)
Anaplastic Lymphoma Kinase/drug effects , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm/genetics , High-Throughput Nucleotide Sequencing/methods , Lung Neoplasms/drug therapy , Precision Medicine , Protein Kinase Inhibitors/therapeutic use , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Circulating Tumor DNA/blood , Humans , Lung Neoplasms/blood , Lung Neoplasms/cerebrospinal fluid , Mutation , Protein Kinase Inhibitors/pharmacologyABSTRACT
Importance: Owing to the improvement of systemic therapies for lung cancer, patients live longer, but the incidence of central nervous system (CNS) metastases also increases. Cerebrospinal fluid (CSF) has been proven better than plasma to reveal unique genetic profiling of intracranial metastases. How genetic alterations in CSF are associated with the prognosis of this heterogeneous patient group remains elusive. Objective: To examine the association of molecular alterations in CSF with the survival of patients with a diagnosis of lung adenocarcinoma and CNS metastases. Design, Setting, and Participants: This retrospective cohort analysis of 94 patients with advanced lung adenocarcinoma and CNS metastases was conducted from July 1, 2016, to July 31, 2018. Patients' CSF samples were collected, and next-generation sequencing of CSF circulating tumor DNA was performed. Main Outcome and Measures: The main outcome was survival after diagnosis with CNS metastases. Genotyping of CSF circulating tumor DNA was studied to examine its association with the clinical outcomes of patients with CNS metastases. Results: Of the 94 patients (49 male; mean [SD] age, 53 [1] years) with lung adenocarcinoma and CNS metastases evaluated, 79 harbored an EGFR variant. The most common genes seen in CSF were EGFR (79 [84.0%]), TP53 (57 [60.6%]), MET (23 [24.5%]), CDKN2A (22 [23.4%]), MYC (20 [21.3%]), NTRK1 (19 [20.2%]), and CDK6 (15 [16.0%]). Cluster analysis identified 5 molecular subtypes of CNS metastases. Patients in cluster I had the shortest median survival after diagnosis of CNS metastases compared with each of the other clusters (cluster I, 7.5 months; cluster II, 55.7 months; cluster III, 17.9 months; cluster IV, 27.9 months; cluster V, 21.0 months) and significantly increased risk of death compared with patients in the other clusters (cluster II: hazard ratio [HR], 4.95; 95% CI, 1.50-16.41; P = .009; cluster III: HR, 4.75; 95% CI, 1.49-15.12; P = .008; cluster IV: HR, 6.38; 95% CI, 1.76-23.09; P = .005; cluster V: HR, 5.42; 95% CI, 1.63-17.98; P = .006). The genetic profiles of cluster I were characterized by a high detection rate of CDK4 (9 of 9 [100%]), TP53 (8 of 9 [88.9%]), MET (7 of 9 [77.8%]), and CDKN2A (7 of 9 [77.8%]). For those with EGFR variants, coalterations with CDK4 (HR, 2.02; 95% CI, 1.03-3.96; P = .04), CDK6 (HR, 2.52; 95% CI, 1.32-4.83; P = .005), and MYC (HR, 2.24; 95% CI, 1.21-4.15; P = .01) were associated with poor outcomes. Conclusions and Relevance: Patients with a diagnosis of lung adenocarcinoma and CNS metastases experienced heterogeneous survival outcomes based on genetic profiling in CSF. These data suggest that CSF might facilitate risk stratifying CNS metastases into appropriate outcomes and provide reference for further clinical study.
Subject(s)
Adenocarcinoma of Lung , Central Nervous System Neoplasms , Circulating Tumor DNA/cerebrospinal fluid , Circulating Tumor DNA/genetics , Lung Neoplasms , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/mortality , Adenocarcinoma of Lung/pathology , Central Nervous System Neoplasms/cerebrospinal fluid , Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/mortality , Central Nervous System Neoplasms/secondary , Female , Genotype , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Male , Middle Aged , Retrospective StudiesABSTRACT
BACKGROUND: This 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). METHODS: A 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. RESULTS: The 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. CONCLUSIONS: This 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.
Subject(s)
Circulating Tumor DNA/genetics , DNA Copy Number Variations , High-Throughput Nucleotide Sequencing , Lung Neoplasms/genetics , Meningeal Neoplasms/cerebrospinal fluid , Mutation , Acrylamides/administration & dosage , Adult , Aged , Aniline Compounds/administration & dosage , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Circulating Tumor DNA/cerebrospinal fluid , Class I Phosphatidylinositol 3-Kinases/genetics , Crown Ethers/administration & dosage , Crown Ethers/adverse effects , Female , Genes, erbB-1 , Humans , Karnofsky Performance Status , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/drug therapy , Male , Meningeal Neoplasms/genetics , Meningeal Neoplasms/secondary , Middle Aged , Mutation Rate , Proto-Oncogene Proteins c-akt/genetics , Quinazolines/administration & dosage , Quinazolines/adverse effects , Young AdultSubject(s)
Adenocarcinoma of Lung/pathology , Biomarkers, Tumor/cerebrospinal fluid , Drug Resistance, Neoplasm , Lung Neoplasms/pathology , Meningeal Carcinomatosis/secondary , Mutation , Acrylamides/therapeutic use , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , Aniline Compounds/therapeutic use , Antineoplastic Agents/therapeutic use , ErbB Receptors/genetics , Female , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Meningeal Carcinomatosis/cerebrospinal fluid , Meningeal Carcinomatosis/drug therapy , Meningeal Carcinomatosis/genetics , Middle Aged , PrognosisABSTRACT
BACKGROUND: Evaluating the molecular characteristics of brain metastases is limited by difficult access and by the blood-brain barrier, which prevents circulating tumor DNA (ctDNA) from entering the blood. In this study, we aimed to compare the sequencing results from cerebrospinal fluid (CSF) ctDNA versus plasma ctDNA, plasma circulating tumor cells (CTCs), and brain tissue specimens from patients with brain metastasis from non-small cell lung cancer (NSCLC). METHODS: This was a prospective study of 21 consecutive patients with NSCLC and brain metastasis diagnosed between April 2018 and January 2019. Samples of CSF and peripheral blood were obtained from all 21 patients. Brain tissues were obtained from five patients after surgical resection. Next-generation sequencing was performed using the Ion system. Single nucleotide variants (SNVs) and small insertions or deletions (indels) were searched. RESULTS: Mutations were detected in the CSF ctDNA of 20 (95.2%) patients. The detection rate of epidermal growth factor receptor (EGFR) mutations in CSF ctDNA was 57.1% (12/21) whereas this rate was only 23.8% (5/21) in peripheral blood ctDNA and in CTCs. EGFR mutations were found in the CSF of 9 of 11 (81.8%) patients with leptomeningeal metastases, as compared with three of 10 (30%) patients with brain parenchymal metastases. Mutations were also detected in KIT, PIK3CA, TP53, SMAD4, ATM, SMARCB1, PTEN, FLT3, GNAS, STK11, MET, CTNNB1, APC, FBXW7, ERBB4, and KDR (all >10%). The status of EGFR and TP53 mutations was consistent between CSF ctDNA and brain lesion tissue in all five patients. CONCLUSION: Sequencing of CSF ctDNA revealed specific mutation patterns in driver genes among patients with NSCLC and brain metastasis. KEY POINTS: In some small-sample studies, the importance of cerebrospinal fluid in guiding the treatment of cancerous brain lesions has been verified in that it may reflect genomic mutations of brain tumors relatively accurately. Cerebrospinal fluid is a new form of liquid biopsy that can be helpful in improving the management of patients with brain metastasis from non-small cell lung cancer by detecting genetic abnormalities specific to brain metastases.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/secondary , Carcinoma, Non-Small-Cell Lung/pathology , Circulating Tumor DNA/genetics , Lung Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Biomarkers, Tumor/cerebrospinal fluid , Brain Neoplasms/cerebrospinal fluid , Brain Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/genetics , Circulating Tumor DNA/cerebrospinal fluid , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Male , Middle Aged , Prognosis , Prospective Studies , Tumor Cells, CulturedABSTRACT
A 47-year-old female with ALK-rearranged lung adenocarcinoma developed leptomeningeal metastasis (LM) after progression on first-line crizotinib. Alectinib 300 mg was commenced and the patient achieved clinical and radiographic improvements. After nine months of alectinib 300 mg, she started to experience symptomatic LM. Two concurrent non-EML4-ALK rearrangements, LOC388942-ALK and LINC00211-ALK, were identified from the CSF but not from the plasma samples. With the primary lung lesions remaining stable, the alectinib dose was increased to 600 mg twice daily which alleviated the clinical symptoms of symptomatic LM. After 7.6 months of alectinib 600 mg, the patient again experienced CNS progression. With both CSF and plasma samples revealing no druggable mutations, the alectinib dose was re-escalated to 900 mg twice daily, resulting in clinical benefits lasting for two months. Her therapy was then switched to lorlatinib which controlled the disease for 8.7 months until her demise. The LINC00211-ALK fusion, which retains the ALK kinase domain, detected from the CSF was the mechanism of treatment efficacy in this patient. The central nervous system (CNS) has been increasingly recognized as a site of treatment failure in multiple cancers, including non-small cell lung cancer (NSCLC). Our case demonstrated that alectinib dose-escalation and lorlatinib overcame ALK inhibitor resistance in the CNS in an ALK-positive LM patient. Furthermore, we provide the first clinical evidence of the efficacy of sequential ALK inhibitors in targeting LINC00211-ALK in a patient with LM.
Subject(s)
Anaplastic Lymphoma Kinase/antagonists & inhibitors , Biomarkers, Tumor/cerebrospinal fluid , Carbazoles/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Cycle Proteins/cerebrospinal fluid , Gene Rearrangement , Meningeal Carcinomatosis/drug therapy , Microtubule-Associated Proteins/cerebrospinal fluid , Piperidines/therapeutic use , Serine Endopeptidases/cerebrospinal fluid , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/metabolism , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle Proteins/genetics , Female , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Meningeal Carcinomatosis/cerebrospinal fluid , Meningeal Carcinomatosis/genetics , Meningeal Carcinomatosis/secondary , Microtubule-Associated Proteins/genetics , Middle Aged , Prognosis , Protein Kinase Inhibitors/therapeutic use , Serine Endopeptidases/geneticsABSTRACT
INTRODUCTION: Dissemination of tumor to the leptomeninges, subarachnoid space, and cerebrospinal fluid (CSF) is termed leptomeningeal metastasis (LM) and occurs in approximately 5% of patients with solid tumors. LM is associated with dismal clinical prognosis, and routine cytologic and radiologic methods for diagnosing LM have limited sensitivity. The CellSearch immunomagnetic rare cell capture assay is FDA-approved to detect circulating tumor cells (CTCs) in peripheral blood, but whether it may have a role in identifying CSF CTCs is still unclear. MATERIAL AND METHODS: CSF specimens from 20 patients with clinically suspected solid tumor LM collected from 2 institutions between October 2016 and January 2019 were evaluated with routine CSF cytology and underwent concurrent CTC testing with the CellSearch assay (Menarini-Silicon Biosystems, Huntingdon Valley, PA). The results of CTC testing were compared to routine CSF cytology and radiologic studies for detecting LM. RESULTS: The CellSearch assay achieved a sensitivity of 88.9% and specificity of 100% for detecting LM (using a threshold of 1 CTC/mL of CSF as the definition of a positive CTC result). One patient with negative CSF cytology but positive CTCs developed positive cytology 37 days later. CONCLUSIONS: In this proof-of-principle pilot study, we demonstrate that the CellSearch assay can be successfully integrated with the routine CSF cytologic workflow to aid in the diagnosis of solid tumor LM. Importantly, CTCs detected by this rare cell capture assay are found in a subset of patients with non-positive routine CSF cytology, which may have significant implications for patient management.
Subject(s)
Adenocarcinoma of Lung/cerebrospinal fluid , Carcinoma, Adenosquamous/cerebrospinal fluid , Carcinoma, Ductal, Breast/cerebrospinal fluid , Cytodiagnosis/methods , Lung Neoplasms/cerebrospinal fluid , Meningeal Carcinomatosis/secondary , Small Cell Lung Carcinoma/cerebrospinal fluid , Triple Negative Breast Neoplasms/cerebrospinal fluid , Adenocarcinoma of Lung/pathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/cerebrospinal fluid , Carcinoma, Adenosquamous/pathology , Carcinoma, Ductal, Breast/pathology , Cohort Studies , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Neoplastic Cells, Circulating , Pilot Projects , Sensitivity and Specificity , Small Cell Lung Carcinoma/pathology , Triple Negative Breast Neoplasms/pathologyABSTRACT
Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non-small-cell lung cancer and melanoma patients. Cell-free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA-Q-PCR or next-generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty-three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine-kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies.
Subject(s)
Acrylamides/administration & dosage , Anaplastic Lymphoma Kinase , Aniline Compounds/administration & dosage , Ascitic Fluid , Lung Neoplasms , Mutation , Neoplasm Proteins , Pleural Effusion, Malignant , Aged , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/metabolism , Ascitic Fluid/metabolism , Ascitic Fluid/pathology , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/cerebrospinal fluid , ErbB Receptors/genetics , Female , HT29 Cells , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasm Proteins/cerebrospinal fluid , Neoplasm Proteins/genetics , Pleural Effusion, Malignant/cerebrospinal fluid , Pleural Effusion, Malignant/drug therapy , Pleural Effusion, Malignant/genetics , Prospective StudiesABSTRACT
BACKGROUND: The presence of specific mutations in the EGFR gene informs the clinical pathway of therapy for patients with lung adenocarcinoma (LAC), including those with central nervous system (CNS) metastases. Plasma circulating cell-free DNA (cfDNA) has been demonstrated to carry the mutational information of LACs, which serves as a biomarker to guide treatment. However, whether the cerebrospinal fluid (CSF) enriches circulating tumor DNA (ctDNA) released from CNS metastatic lesions of LAC, and whether the CSF ctDNA can be used to characterize these lesions remains unknown. OBJECTIVE: To explore the EGFR status in CNS metastases of LAC patients, and to guide the treatment of intra- and extracranial tumors in these patients. PATIENTS AND METHODS: The EGFR mutational status in the cfDNA from paired CSF and plasma samples from LAC patients with CNS metastases, including 20 brain metastases (BM) and 15 leptomeningeal metastases (LM), was assessed by droplet digital polymerase chain reaction (ddPCR). The clinical outcomes of the EGFR status-based intervention were investigated. RESULTS: EGFR mutations were detected in 23/35 LAC patients (65.7%). EGFR mutations in the plasma or CSF were detected in 6/11 (54.5%) and 5/10 (50%) BM patients, and in 4/11 (36.4%) and 9/12(75%) LM patients, respectively. The prevalence of the T790M mutation was significantly higher in plasma (9/23) than in CSF (3/23) samples. The sensitivity and specificity of the ddPCR-based EGFR mutation test in CSF or plasma samples versus the primary tumor samples were 56% and 89% versus 46% and 100%, respectively. Twelve patients received a first-generation EGFR TKI (tyrosine kinase inhibitor) after the detection of sensitive EGFR mutations in their CSF or plasma, and five patients were switched from a first-generation EGFR TKI to osimertinib after the detection of the T790M mutation. CONCLUSIONS: The EGFR T790M mutation in plasma cfDNA is a sensitive marker for EGFR TKI resistance when CNS metastases progressed. CSF ctDNA increases the diagnostic validity for EGFR genotyping of lung cancer brain metastasis. ddPCR in CSF and plasma samples could provide less invasive and close monitoring of the EGFR status of LAC patients with CNS metastases.
Subject(s)
Adenocarcinoma of Lung/pathology , Biomarkers, Tumor/analysis , Carcinoma, Non-Small-Cell Lung/pathology , Central Nervous System Neoplasms/secondary , Circulating Tumor DNA/blood , Mutation , Polymerase Chain Reaction/methods , Adenocarcinoma of Lung/blood , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/genetics , Adult , Aged , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/genetics , Central Nervous System Neoplasms/blood , Central Nervous System Neoplasms/cerebrospinal fluid , Central Nervous System Neoplasms/genetics , ErbB Receptors/genetics , Female , Follow-Up Studies , Humans , Lung Neoplasms/blood , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Middle Aged , Prognosis , Protein Kinase Inhibitors/therapeutic use , Retrospective Studies , Survival RateABSTRACT
BACKGROUND: The cobas® epidermal growth factor receptor (EGFR) Mutation Test v2 designed for cell-free DNA (cfDNA) is approved as a companion diagnostic for osimertinib therapy. The aim of this study was to evaluate the concordance of EGFR mutation detection between paired primary or recurrent samples, and cerebrospinal fluid (CSF) cytology samples of lung cancer patients. METHODS: In total, 26 lung cancer patients with supernatant cytology cfDNA in CSF were analysed for EGFR mutations using the cobas® EGFR Mutation Test v2.0 designed for cfDNA, and the concordance rates between CSF cfDNA and primary or recurrent samples were investigated. RESULTS: Of the 26 CSF cytology cfDNA samples, 46.1% (12/26) were valid and 53.9% (14/26) were invalid. Sensitivity, specificity and accuracy between the valid CSF cfDNA samples and primary or recurrent samples for detection of EGFR mutation, including T790M were 87.5%, 100.0% and 91.7%, respectively. Amounts of both inflammatory cells and tumour cells in CSF cytology were higher in the valid evaluation samples than in the invalid samples (P < .05), and mutant EGFR was detected in 80.0% (4/5) of the valid CSF cytology cfDNA samples with a negative cytology diagnosis. CONCLUSIONS: The cobas® EGFR Mutation Test v2.0 can accurately detect EGFR mutations, including T790M, from supernatant cfDNA of CSF cytology samples. Utilisation of supernatant cytology cfDNA in CSF will allow us to perform both EGFR mutation analysis and cytopathological diagnosis at the same time. This represents a new role of cytology in patient treatment, based on assured sample quality.
Subject(s)
Cell-Free Nucleic Acids/cerebrospinal fluid , Cytodiagnosis , Lung Neoplasms/cerebrospinal fluid , Aged , Aged, 80 and over , ErbB Receptors/cerebrospinal fluid , ErbB Receptors/genetics , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Mutation/geneticsABSTRACT
BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are associated with leptomeningeal metastases (LM) of nonsmall cell lung cancer and sensitivity to tyrosine kinase inhibitor (TKI) treatment. Owing to the difficulty of obtaining carcinomatous meningeal tissue for analysis, cerebrospinal fluid (CSF) might be an alternative. OBJECTIVE: To investigate the EGFR mutation detection in the CSF of lung adenocarcinoma patients with LM. METHODS: Twenty-five lung adenocarcinoma patients with LM diagnosed by CSF cytology were retrospectively evaluated. The results of EGFR mutation detection in CSF, the treatment plan, and clinical outcome information were recorded. RESULTS: Nineteen patients had a known EGFR status in their primary tumors. Twenty patients received EGFR mutation analysis in CSF after LM diagnosis and 14 of them with a known EGFR mutation status of both primary tumors and CSF. Ten (71.4%) had the same EGFR gene status. In primary tumors, no T790M mutations were detected, whereas in CSF, 2 L858R cases and 1 19del case had T790M mutations at the same time. The detection rate of T790M mutations in CSF was 18.1% (2 of 11) in all cases with EGFR-sensitive mutations in the primary lesion. CONCLUSIONS: EGFR mutation detection in CSF of lung adenocarcinoma patients with LM might be an alternative when leptomeningeal biopsy cannot be applied and may help to guide TKI treatments.
Subject(s)
Adenocarcinoma of Lung/genetics , Cerebrospinal Fluid/chemistry , Lung Neoplasms/genetics , Meningeal Carcinomatosis/secondary , Mutation , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/enzymology , Adenocarcinoma of Lung/pathology , ErbB Receptors/cerebrospinal fluid , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Male , Meningeal Carcinomatosis/cerebrospinal fluid , Meningeal Carcinomatosis/enzymology , Meningeal Carcinomatosis/genetics , Middle Aged , Neoplasm Metastasis , Retrospective Studies , Treatment OutcomeABSTRACT
BACKGROUND: Leptomeningeal metastases (LM) are much more frequent in patients of non-small lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFRTKI) shows promising efficacy for LM. OBJECTIVE: The aim of this study was to analyze the concentration of osimertinib and gene variation of circulating tumor DNA (ctDNA) in human plasma and cerebrospinal fluid (CSF). Furthermore, we explored whether ctDNA in CSF might be used as a biomarker to predict and monitor therapeutic responses. METHODS: The dynamic paired CSF and blood samples were collected from the NSCLC patient with LM acquired EGFR-TKI resistance. A method based on ultra-high performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) was developed and validated for detecting osimertinib in CSF and plasma samples. Gene variations of ctDNA were tested by next-generation sequencing with a panel of 1021 genes. RESULTS: The concentrations of osimertinib in CSF were significantly lower than that in plasma (penetration rate was 1.47%). Mutations included mTOR, EGFR, CHECK1, ABCC11, and TP53 were explored in ctDNA from plasma and CSF samples. The detected mutation rate of CSF samples was higher than that of plasma samples (50% vs. 25%). Our data further revealed that the variations allele frequency (VAF) and molecular tumor burden index (mTBI) of ctDNA derived from CSF exhibited the negative correlation with efficacy of treatment. CONCLUSION: ctDNA from CSF might be a useful biomarker for monitoring the efficacy of treatment and an effective complement to nuclear magnetic resonance imaging (MRI) for LM.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Circulating Tumor DNA/blood , Meningeal Neoplasms/secondary , Mutation , Acrylamides/blood , Acrylamides/cerebrospinal fluid , Acrylamides/therapeutic use , Adenocarcinoma of Lung/blood , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Aniline Compounds/blood , Aniline Compounds/cerebrospinal fluid , Aniline Compounds/therapeutic use , Antineoplastic Agents/blood , Antineoplastic Agents/cerebrospinal fluid , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/analysis , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/cerebrospinal fluid , Carcinoma, Non-Small-Cell Lung/drug therapy , Circulating Tumor DNA/genetics , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/cerebrospinal fluid , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Meningeal Neoplasms/blood , Meningeal Neoplasms/cerebrospinal fluid , Meningeal Neoplasms/drug therapy , Middle Aged , PrognosisABSTRACT
The central nervous system is a common site of relapse in patients receiving crizotinib, which is presumed to be associated with the low concentration of crizotinib in the cerebrospinal fluid (CSF). Our patient received surgical treatment for anaplastic lymphoma kinase-positive stage IIA lung adenocarcinoma. His cancer recurred with brain metastases and carcinomatous meningitis. We started whole-brain radiation therapy (WBRT) and subsequently administered crizotinib. The concentration of crizotinib on day 15 in the plasma was 158 ng/mL, and that in the spinal fluid was 4.32 ng/mL. WBRT may elevate the CSF/plasma crizotinib concentration ratio; clinicians may therefore consider performing WBRT prior to crizotinib initiation.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents/cerebrospinal fluid , Crizotinib/cerebrospinal fluid , Lung Neoplasms/drug therapy , Meningeal Carcinomatosis/drug therapy , Adenocarcinoma of Lung/cerebrospinal fluid , Adenocarcinoma of Lung/radiotherapy , Adenocarcinoma of Lung/secondary , Anaplastic Lymphoma Kinase/analysis , Antineoplastic Agents/therapeutic use , Brain Neoplasms/cerebrospinal fluid , Brain Neoplasms/radiotherapy , Brain Neoplasms/secondary , Combined Modality Therapy , Cranial Irradiation , Crizotinib/therapeutic use , Disease Progression , Fatal Outcome , Humans , Lung Neoplasms/cerebrospinal fluid , Male , Meningeal Carcinomatosis/cerebrospinal fluid , Meningeal Carcinomatosis/radiotherapy , Middle Aged , Neoplasm Recurrence, Local/cerebrospinal fluid , Neoplasm Recurrence, Local/drug therapyABSTRACT
BACKGROUND: 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.