HER2 exon 20 insertion mutations in lung adenocarcinoma with leptomeningeal metastasis: a case report and response to poziotinib.

Leptomeningeal metastasis (LM) is associated with poor prognosis and represents a terminal event of non-small cell lung cancers (NSCLC). In previous studies, most of LM-patients have detected epidermal growth factor receptor (EGFR) mutation and responded to the third generation of EGFR-tyrosine kinase inhibitor (TKI). This study aimed to report a case of ERBB2 (HER2) exon 20 insertion mutations in the cerebrospinal fluid (CSF) of LM-patient which response to poziotinib. At the beginning, postoperative pathology showed a primary invasive adenocarcinoma with no mutations in EGFR and ROS-1. Pemetrexed plus carboplatin combined with bevacizumab was administered as the first-line followed by bevacizumab alone for continuation maintenance therapy. Targeted therapy and immunotherapy were given after the disease progressed in two months. Subsequently, the patient developed mental symptoms and adenocarcinoma cells were found in the CSF. Next-generation sequencing (NGS) results showed HER2 exon 20 insertion mutations in the primary tissue, CSF and plasma samples. Then, poziotinib was administered and the symptoms improved significantly after 3 days and the progress free survival was nearly 2 months. Therefore, we speculate that the CSF concentration and penetration rate of poziotinib may significantly higher than of other TKIs so that it achieves a higher CSF concentration than standard dosing, and successfully controlled LM. It may provide a new therapeutic option for LM-patient and may be especially who are lung adenocarcinoma with HER2 exon 20 insertion.

Genomic comparison between cerebrospinal fluid and primary tumor revealed the genetic events associated with brain metastasis in lung adenocarcinoma.

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.

[Different Gene Mutation Spectrum of the Paired CSF and Plasma Samples in Lung Adenocarcinoma with Leptomeningeal Metastases: the Liquid Biopsy Based on Circulating Tumor DNA].

BACKGROUND: Leptomeningeal metastasis (LM) are a severe complication of non-small cell lung cancer (NSCLC), and normally accompanied by poor prognosis. For the patients with targetable mutations, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the preferred treatment, but the acquired TKI resistance is inextricable. The aim of this study is to analyze the different gene mutation spectrum and mutation frequency of the cerebrospinal fluid (CSF) and plasma in NSCLC patients with LM, and screen out the drug-resistant mutations so as to guide the choice of treatment accurately. METHODS: The paired CSF and plasma samples were collected from the NSCLC-LM patients with acquired TKI resistance. Next generation sequencing (NGS) was used to detect the gene variations of circulating tumor DNA (ctDNA). RESULTS: A total of 18 NSCLC patients with LM were collected. Of the basic mutations, 11 cases (61.11%) were EGFR, 6 cases (33.33%) were anaplastic lymphoma kinase (ALK), and 1 case (5.56%) was ROS proto-oncogene 1, receptor tyrosine kinase (ROS1). Tumor protein p53 gene (TP53) and mesenchymal-epithelial transition factor (MET) were the two most frequently accompanying mutated genes in CSF ctDNA. The detected mutation rate of CSF samples was higher than that of plasma samples (100.00% vs 66.67%, P=0.006), and the maximum allelic fractions were all higher in CSF than in plasma (P<0.001). Abundant single-nucleotide variations (SNV) and copy number variants (CNV) were detected in CSF, the amount of both of which were more than in blood. In addition, the CSF and plasma samples of patients treated with several TKIs had more SNV mutations than patients who received only a single TKI treatment. CONCLUSIONS: For the patients of NSCLC, ctDNA in CSF could reveal genomic alterations of LM more exactly and overally than it in plasma, thus could be an optimal source of liquid biopsy for guiding therapy, monitoring therapeutic effect, and predicting prognosis.

Association of Cerebrospinal Fluid Tumor DNA Genotyping With Survival Among Patients With Lung Adenocarcinoma and Central Nervous System Metastases.

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.

Integration of rare cell capture technology into cytologic evaluation of cerebrospinal fluid specimens from patients with solid tumors and suspected leptomeningeal metastasis.

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.

Simultaneous primary cancer occurrence of melanoma and pulmonary adenocarcinoma in leptomeningeal metastases: a case report.

BACKGROUND: Leptomeningeal metastasis (LM) is a predominantly late stage, devastating complication of a variety of malignant solid tumors. Diagnosis relies predominantly on neurological, radiographic, and cerebrospinal fluid (CSF) assessments. Recently, liquid biopsy tests derived from CSF has shown to be a feasible, noninvasive promising approach to tumor molecular profiling for proper brain cancer diagnostic treatment, thereby providing an opportunity for CSF-based personalized medicine. However, LM is typically misleadingly assumed to originate from only one primary tumor type. CASE PRESENTATION: In this case report, we provide first evidence of the co-occurrence of LM originating from more than one primary tumor types. DISCUSSION AND CONCLUSIONS: Based on this patient case profile, the co-occurrence of LM from two or more primary tumor types should be accounted for when deriving diagnostic conclusions from liquid biopsy tests.

Digital PCR-Based Detection of EGFR Mutations in Paired Plasma and CSF Samples of Lung Adenocarcinoma Patients with Central Nervous System Metastases.

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.

Epidermal Growth Factor Receptor Mutation Detection in Cerebrospinal Fluid of Lung Adenocarcinoma Patients with Leptomeningeal Metastasis.

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.

A Low Crizotinib Concentration in the Cerebrospinal Fluid Causes Ineffective Treatment of Anaplastic Lymphoma Kinase-positive Non-small Cell Lung Cancer with Carcinomatous Meningitis.

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.

Osimertinib Quantitative and Gene Variation Analyses in Cerebrospinal Fluid and Plasma of a Non-small Cell Lung Cancer Patient with Leptomeningeal Metastases.

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.

Detection of EGFR, KRAS and BRAF mutations in metastatic cells from cerebrospinal fluid.

BACKGROUND: In lung adenocarcinoma, molecular profiling of actionable genes has become essential to set up targeted therapies. However, the feasibility and the relevance of molecular profiling from the cerebrospinal fluid (CSF) in the context of meningeal metastasis have been poorly assessed. METHODS: We selected patients with stage IV lung adenocarcinoma harbouring metastatic cells in the CSF after cytological analysis. Seven samples from six patients were eligible for molecular testing of epidermal growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS), v-Raf murine sarcoma viral oncogene homologue B1 (BRAF) and human epidermal growth factor receptor 2 (HER2) mutations using quantitative polymerase chain reaction (PCR) high-resolution melting curve analysis and Sanger sequencing after DNA extraction from the cell pellets of the CSF. RESULTS: Five patients showed mutations in one or two actionable genes, two harboured an EGFR mutation (exons 19 and 21), one only a KRAS mutation, one both EGFR and KRAS mutations and one a BRAF mutation. In all cases, the results of mutation testing provided new major information for patient management, leading to therapeutic adaptation. CSF molecular analysis identified mutations not detected in other neoplastic sites for two patients. In one case, the EGFR p.Thr790Met was identified. CSF was also the only sample available for genetic testing for almost all patients at the time of disease progression. CONCLUSIONS: When cancer cells are present in the CSF, the molecular profiling from the cell pellets is relevant, as it can detect supplemental or different mutations compared to a previous analysis of the primitive tumour or plasma cell-free DNA and allows the adaptation of the treatment strategy.