Study On The Neoplastic Meningitis Based On The Next-generation Sequencing Of Cerebrospinal Fluid

Neoplastic meningitis(NM)aslo called meningeal carcinomatosis(MC),refers to the dissemination of malignant cells to the leptomeninges,arachnoid mater,cerebrospinal fluid(CSF),and pia mater,which occurs in approximately 5% to 8% of all patients with cancer.According to the 2018 global cancer report,there will be 18.1 million new cancer cases and 9.6million cancer deaths worldwide,with nearly half of new cancer cases and more than half of cancer deaths occurring in Asia.Since 2010,cancer has become the leading cause of death in China.According to China’s latest cancer report statistics released by the National Cancer Center in 2019,lung cancer ranks first in the incidence of malignant tumors in China.Brain is the most common distant metastasis site of lung cancer.Leptomeningeal metastases(LM)occur in 3%～5% of non-small cell lung cancer(NSCLC)patients.NM causes significant morbidity and mortality.The early diagnosis and timely treatment are likely to have the greatest impact on improving outcome.However,NM is easily to be missed in diagnosis and easily misdiagnosed because of diverse clinical manifestations and the lack of sensitive and specific diagnostic tools,which has presented difficulties for early treatment of patients with NM.Currently,the diagnosis of NM is routinely confirmed on the basis of clinical signs and symptoms,CSF cytology,and/or neuroimaging [contrast-enhanced brain magnetic resonance imaging(MRI)and/or computed tomography(CT)] findings.The presence of neoplastic cells in the CSF is the most useful finding to confirm the diagnosis and CSF cytology remains the gold diagnostic standard,but 25～30% of suspected clinical cases with NM diagnosed based on clinical picture and neuroimaging findings will not be confirmed by this method.Although the positive rate of CSF cytology for neoplastic cells will increase with thenumber of punctures,the false negative rate is still high,due to the similarity between tumor cells and ependymal cells,contamination by blood brought about by multiple punctures,difficulty of the distinction lymphoma cells caused by viral infections of central nervous system(CNS),few tumor cells in the collected specimen and inadequate preparation of the sample.Contrast-enhanced brain MRI and/or CT is the technique of choice to evaluate patients with suspected NM but still has a 30% incidence of false-negative results.Therefore,it is important to evaluate an alternative method for diagnosis of NM especially for cases with persistently negative CSF cytology and/or persistently negative neuroimaging results.As a new cancer detection technology,compared with traditional tissue biopsy,the advantage of liquid biopsy is that it is minimally invasive,and can be serially repeated,thus allowing extracting information from the cancer in real time and overcoming tumor heterogeneity.Due to the difficulty to obtain tissue of NM,and the poorly understanding of pathogenesis and resistance mechanism of NM,more and more attention has been paid to explore the use of plasma and cerebrospinal fluid to track the gene evolution of tumor cells.The circulating tumor DNA(ctDNA)of plasma has been applied to detect multiple different types of cancer.However,plasma ctDNA from tumors confined to central nervous system was infrequently detectable given that physical obstacles such the blood-brain barrier could prevent ctDNA from entering the blood circulation.Given to NM disseminates over the leptomeningeal surface,neoplastic cells shed into CSF and CSF is in direct contact with neoplastic cells and meninges,CSF is clearly a potential source of fluid biopsy sample for NM and to analyze the genomic level of the primary and metastatic lesions of NM.Many studies have shown that CSF ctDNA could be an important method of liquid biopsy in patients with CNS cancers.However,only a single tumor type or single primary tumor type was included in these studies.Given that NM is involvement of the leptomeninges by metastatic tumors and can be observed in various kinds of solid tumors,we hope to evaluate the amounts of ctDNA among different primary tumor typesand evaluate the clinical value of CSF ctDNA as liquid biopsy medium in the diagnosis of NM.NM occurs in 3%～5% of NSCLC patients,especially in 9.4% of those with an epidermal growth factor receptor(EGFR)mutation.The incidence of NM may increase with better treatments and the overall longer survival of patients with cancer in general.EGFR tyrosine kinase inhibitor(TKI)markedly prolong survival,but NM is associated with very poor survival.Though EGFR TKI targeted therapy had been described as an independent predictor of favorable survival in patients with NM,gene status were based on primary cancer tissue rather than CSF.EGFR TKI therapy for NM was found to prolong survival,but the impact of CSF EGFR mutations was not clear because of the small number of patients who underwent EGFR gene testing.The heterogeneity between primary tumors and metastatic tumors of the central nervous system highlights the importance of CSF ctDNA sequencing for metastatic tumors of the central nervous system.Several studies have shown that the response rate of EGFR TKI treatment can be as high as70%～80% in NSCLC patients with EGFR gene mutation.However,due to the limitation of blood-brain barrier,the effect of EGFR TKI on meningeal metastasis is weakened.The concentration of CSF treated with standard dose of EGFR TKI per day is not enough to achieve the effect of treating NM.Therefore,it is necessary to evaluate the choice of EGFR TKI treatment after the diagnosis of NM and its clinical effect and impact on the overall survival.Accordingly,the purpose of the current study is to explore the value of CSF ctDNA as a liquid biopsy in patients with NM secondary to different types of cancer,and to analyze the cancer-related gene mutations in CSF ctDNA samples from NM patients and the potential mechanism of meningeal metastasis of malignant tumor cells.To evaluate the clinical efficacy and prognostic significance of different EGFR TKI regimens in NM patients with CSF EGFR positive.Part one Evaluating the cerebrospinal fluid ctDNA detection by next-generation sequencing in the diagnosis of neoplastic meningitisObjective: Neoplastic meningitis(NM)is the most severe form of brain metastasis and causes significant morbidity and mortality.Currently,the diagnosis of NM is routinely confirmed on the basis of clinical manifestation,positive cerebrospinal fluid(CSF)cytology,and/or neuroimaging features.However,negative rate of CSF cytology and neuroimaging findings often result in a failure to diagnose NM from the patients who actually have the disease.Here we evaluate the sensitivity of CSF circulating tumor DNA(ctDNA)as liquid biopsy medium in the diagnosis of NM patients.Methods: In total,35 confirmed NM patients who underwent lumbar puncture for CSF cytology examination,CSF ctDNA extraction and cancer-associated gene mutations detection by next-generation sequencing(NGS)and underwent contrast-enhanced brain MRI and/or CT between October 2014 and September 2017 were enrolled in this study.The sensitivity of CSF cytology,CSF ctDNA and neuroimaging were compared in the diagnosis of NM.Results: The most frequent primary tumor in this study was lung cancer(26/35,74%),followed by gastric cancer(2/35,6%),breast cancer(2/35,6%),prostatic cancer(1/35,3%),parotid gland carcinoma(1/35,3%)and lymphoma(1/35,3%)while no primary tumor could be found in the remaining 2 patients in spite of using various inspection methods.Twenty-five CSF samples(25/35;71%)were found neoplastic cells in CSF cytology examination while all of the 35 CSF samples(35/35;100%)were revealed having detectable ctDNA in which cancer-associated gene mutations were detected.All of 35 patients with NM in the study underwent contrast-enhanced brain MRI and/or CT and 22 neuroimaging features(22/35;63%)were consistent with MC.The sensitivity of the neuroimaging was 88%(95% confidence intervals [95% CI],75 to 100)(P = 22/25)and 63%(95% CI,47 to 79)(P = 22/35)compared to those of CSF cytology and CSF ctDNA,respectively.The sensitivity of the CSF cytology was 71%(95% CI,56 to 86)(P = 25/35)compared to that of CSF ctDNA.Conclusions: This study suggests a higher sensitivity of CSF ctDNA than those of CSF cytology and neuroimaging findings.We find cancer-associated gene mutations in ctDNA from CSF of patients with NM at100% of our cohort,and utilizing CSF ctDNA as liquid biopsy technology based on the detection of cancer-associated gene mutations may give additional information to diagnose NM with negative CSF cytology and/or negative neuroimaging findings.Part two Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next-generation sequencingObjective: We aimed to profile the somatic gene mutations and the copy number variation(CNV)in CSF ctDNA from patients with NM and to analyze cancer-associated gene mutations in CSF ctDNA samples and the potential mechanism of meningeal metastasis of malignant tumor cells.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: In this study,62 CSF samples from 58 patients with NM were analyzed.There were 27 male and 31 female.The most frequent primary tumor in this study was lung cancer(42/58,72.4%),followed by gastric cancer(4/58,6.9%),breast cancer(3/58,5.2%),rectal cancer(2/58,3.4%),prostatic cancer(1/58,1.7%),parotid gland carcinoma(1/58,1.7%),lymphoma(1/58,1.7%)and glioblastoma(1/58,1.7%)while no primary tumor could be found in the remaining 3 patients in spite of using various inspection methods.Furthermore,among the 62 CSF samples,30 CSF samples were collected from 28 patients who received intrathecal chemotherapy and systemic therapy(systemic chemotherapy,radiotherapy,and/or molecule-targeted therapy).11 CSF samples were obtained from 11 patients who received intrathecal chemotherapy without systemic therapy.12 CSF samples were obtained from 12 patients who received systemic therapy without intrathecal chemotherapy.The remaining nine CSF samples were collected from nine patients who did not receive any anticancer therapy.Cancer-associated gene mutations occurred in all 62 CSF ctDNA samples.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 CNV 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 cancer-associated gene mutations in all CSF ctDNA samples and these mutated genes might affect meningeal metastasis through PI3K-Akt signaling pathway.Part three The clinical and prognostic significance of EGFR TKI for CSF EGFR positive NM patients with lung cancerObjective: We aimed to evaluate clinical efficacy of EGFR TKI for CSF EGFR positive NM patients with lung cancer and analyze the prognostic factors.Methods: We retrospectively reviewed the data of NM patients with lung cancer who had CSF EGFR driver mutations between October 2014 and February 2020 at the second Hospital of Hebei Medical University.Results: Of the 44 NM patients with lung cancer who had CSF EGFR driver mutations,17 were male(38.6%),the median age was 57 years(range,32-74 years),and 27(61.4%)had an ECOG performance status(PS)≥3.Thepatients were diagnosed with NM after a median of 14.0 months(range,0–87.3 months)from the initial diagnosis of lung cancer.NM was present in11 patients at the time of initial diagnosis.The median concentration of CSF circulating tumor DNA(ctDNA)was 3.1 ng/ul(range,0.144-30.6 ng/ul).The clinical remission rate in patients who received EGFR TKI after diagnosis with NM was higher than those who didn’t(73.3% vs.21.4%,P = 0.001).The clinical remission rate in patients who initiated EGFR TKI,switched EGFR TKI treatments and received high-dose EGFR TKI treatment was higher than those who continued with their current EGFR TKI treatment and not administered EGFR TKI treatment after NM diagnosis(77.8% vs.23.5%months,P < 0.001).The clinical remission rate of with and without Osimertinib treatment after NM diagnosis was 75.0% and 41.7%,respectively,and with significant difference(P = 0.026).The median overall survival(OS)after the diagnosis of NM was 16.0 months(95% CI,2.0-30.0 months).The median OS in patients with an ECOG PS of 1-2 was longer than patients with an ECOG PS of 3-4(32.6 vs.10.6 months,P<0.001).The median OS in patients with EGFR TKI treatment was longer than patients without EGFR TKI treatment before NM diagnosis(32.6 vs.12.8 months,P = 0.032).The median OS in patients with EGFR TKI treatment was longer than patients without EGFR TKI treatment after NM diagnosis(31.4 vs.5.4 months,P <0.001).The median OS in patients who initiated EGFR TKI,switched EGFR TKI treatments and received high-dose EGFR TKI treatment was longer than those who continued with their current EGFR TKI treatment and not administered EGFR TKI treatment after NM diagnosis(29.6 vs.8.9 months,P= 0.001).The median OS in patients who received Osimertinib treatment was longer than those who didn’t received Osimertinib treatment after NM diagnosis(32.6 vs.11.1 months,P = 0.004).Univariate analysis by the Kaplan–Meier test showed that ECOG PS,EGFR TKI treatment before NM diagnosis,EGFR TKI treatment after NM diagnosis,the option of EGFR TKI treatment after NM diagnosis and Osimertinib treatment after NM diagnosis were correlated with prognosis of patients with NM.Multivariate analysissuggested that initiated,switched and received high-dose EGFR TKI treatment in NM patients with CSF EGFR positive was independent predictor for longer survival.Conclusions:1.NM patients treated with EGFR TKI had higher clinical remission rate and longer median overall survival than those without EGFR TKI.2.NM patients who initiated EGFR TKI,switched EGFR TKI and received high-dose EGFR TKI had higher clinical remission rate and longer median overall survival than those who continued with their current EGFR TKI and not administered EGFR TKI after NM diagnosis.3.NM patients who received Osimertinib treatment had higher clinical remission rate and longer median overall survival than those who didn’t received Osimertinib treatment after NM diagnosis.4.Initiated,switched and received high-dose EGFR TKI treatment in NM patients with CSF EGFR positive is associated with prolong survival period.