Advancing Non-Small Cell Lung Cancer Treatment: A Case Series on Pemetrexed Intrathecal Chemotherapy Monitored Through Circulating Tumor DNA in Cerebrospinal Fluid

Advancing Non-Small Cell Lung Cancer Treatment: A Case Series on Pemetrexed Intrathecal Chemotherapy Monitored Through Circulating Tumor DNA in Cerebrospinal Fluid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Advancing Non-Small Cell Lung Cancer Treatment: A Case Series on Pemetrexed Intrathecal Chemotherapy Monitored Through Circulating Tumor DNA in Cerebrospinal Fluid Weiping Hong, Lei Wen, Yanying Yang, Hui Wang, Qingjun Hu, Chuqiao Liang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4482413/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Intrathecal pemetrexed (IP) treatment represents a promising approach for managing leptomeningeal metastasis (LM) in cancer patients. However, a standardized and measurable method to evaluate the efficacy of IP for non-small cell lung cancer (NSCLC) patients with LM is currently lacking. This report describes three NSCLC-LM cases treated with IP following progression with tyrosine kinase inhibitors (TKIs) alone. We observed their responses through next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF). The outcomes were favorable for patient 1 and 2, whereas patient 3 experienced a relapse. Notably, changes in the allele frequency (AF) of ctDNA mutations corresponded with clinical outcomes across these patients, which were also corroborated by multiple traditional clinical markers. This observation is significant, particularly in patient 3, where ctDNA monitoring effectively described the patient's temporary improvement followed by deterioration. Furthermore, in patient 2, the transition from a positive to a negative cytological test, alongside persistent positive NGS results, underscores the higher sensitivity of NGS compared to conventional cytological analysis. This suggests that longitudinal ctDNA monitoring using CSF samples may serve as an effective and independent method for assessing and dynamically tracking the response to IP treatment in NSCLC-LM patients. This approach has the potential to refine therapeutic strategies and improve patient outcomes. Figures Figure 1 Figure 2 Figure 3 Introduction Approximately 5% of patients with advanced non-small cell lung cancer (NSCLC) may develop leptomeningeal metastasis (LM), which is more prevalent in those with EGFR mutations. (Remon et al. 2017 , Liao et al. 2015 )Patients with LM and a low performance status (PS) may have a poor prognosis; the median overall survival (OS) of LM NSCLC patients is only 3.6 to 11 months(Liao and Lee 2015, Umemura et al. 2012 ). Although targeted therapies, particularly tyrosine kinase inhibitors (TKIs), are effective against primary tumors, the blood‒brain barrier (BBB) may limit intracranial performance, resulting in variable patient survival outcomes. (Cheng et al. 2018) LM patients have been shown to benefit from a variety of treatments, including surgery, radiation, systematic chemotherapy, targeted chemotherapy, immunotherapy, and intrathecal injection, but there is still a lack of conventional treatment paradigms for LM patients. In patients with NSCLC, intrathecal administration, which delivers medications directly into the subarachnoid space via cerebrospinal fluid (CSF), was reported to be highly effective in controlling LM. (Roguski et al. 2015 , Pan et al. 2016 ) A recent clinical trial has proven the efficacy and safety of intrathecal pemetrexed combined with dexamethasone for treating TKI-failed LM EGFR + NSCLC. At the same time, they revealed the challenge of their study on measuring treatment responses.(Fan et al. 2021 ) Evaluating the efficacy of treatment for LM remains difficult and lacks standardization. Due to the diffusion of LM lesions, it is challenging to quantify tumor size using imaging techniques. The Karnofsky performance score (KPS) is a commonly used tool for assessing functional impairment in LM patients (Schag et al. 1984 ), but it assesses treatment efficacy indirectly through patient performance and cannot reveal tumor changes in a quantifiable manner. Response Assessment in Neuro-Oncology (RANO) is a generally accepted response criterion that evaluates treatment response by incorporating a novel radiographic scorecard, CSF cytological examination or flow cytometry, and neurological examination, but it is limited in its ability to measure lesions for response assessment. (Chamberlain et al. 2017 , Le Rhun et al. 2019 ) On the other hand, the significant issue of high false negatives in the detection of cerebrospinal fluid cytology also poses a major challenge in monitoring treatment effectiveness.(Hyun et al. 2016) Recent research has demonstrated that circulating tumor DNA (ctDNA) released by tumor apoptosis can be used as a biomarker to track treatment responses and tumor evolution. (Diehl et al. 2008 ) CtDNA from cerebrospinal fluid (CSF) has also demonstrated superior intracranial response prediction performance compared to that from plasma. (Li et al. 2022 ) The urgent need for standardization of intrathecal chemotherapy, a novel and effective treatment for patients with LM, is inextricably linked to the establishment of quantifiable evaluation methods. In this study, we employed Next-generation sequencing (NGS) to identify ctDNA in CSF from three patients diagnosed with LM. These patients received intrathecal pemetrexed (IP) therapy, which was administered as an initial single dose of 20 mg in week 1, followed by 30 mg in week 3. Subsequently, the patients continued to receive doses of 30 mg to 50 mg once every three weeks, as indicated by changes in the NGS allele frequency (AF) until disease progression or intolerance occurred. To the best of our knowledge, this is the first report on therapeutic responses and clinical observations of this approach. Patient 1 In October 2022, a 66-year-old female patient who had been treated with surgery and EGFR-TKIs for lung adenocarcinoma metastases since 2016 (Fig. 1 A) visited our hospital due to numbness in her left hand. Her mental state, appetite, and sleep were all normal, yet she was experiencing numbness in her limbs, particularly in her left hand, which significantly affected her ability to perform daily self-care tasks (KPS 50). In addition to positive cytology of the CFS, MRI revealed a new lesion on her meninges, leading to a diagnosis of lung adenocarcinoma meningeal metastases. After the failure of targeted treatments, between October 2022 and March 2023, the patient underwent six IP treatments, with an initial intracranial pressure (ICP) of 210 mmHg for the first time. The collection of ctDNA from her CSF began prior to receiving the third IP. Along with significant symptomatic improvement, we observed a reduction in the mutation allele frequency (AF) of ctDNA in both the cerebrospinal fluid (CSF) and intracranial pressure (ICP) throughout the treatment (Fig. 1 B). Specifically, the mean AF decreased from 51.94% (ranging from 19.42–90.49%) to 19.39% (ranging from 7.97–43.22%). Her capacity to walk, engage in verbal communication, and maneuver her limbs was enhanced, allowing her to regain her self-care abilities (KPS 80). A general dose (30 mg) of pemetrexed was given, as both NGS and clinical response improved. Retrospectively, the EGFR L858R mutation was identified by NGS testing after surgical treatment of metastatic front parietal intra-axial brain tumors in 2016. She was treated with gefitinib for more than two years and then switched to osimertinib in 2019 due to the slow progression of her brain metastases. Gamma-knife surgery was performed for brain tumors in 2020. Although the patient had been taking osimertinib for twenty-four months without experiencing headache or dizziness, limb numbness caused by meningeal metastases did not improve. The local IP treatment relieved numbness in her limbs, which was observed through the decrease in ctDNA AF in CSF, along with improvements in KPS and ICP. From the start of the intrathecal pemetrexed treatment until the last follow-up in May 2023, the patient had a progression-free survival (PFS) of 6 months and an overall survival (OS) of 6 months. Patient 2 A 66-year-old male patient who had a 30-year history of smoking, diagnosed with moderately differentiated adenocarcinoma with metastases in the lungs, lymph nodes, bones and nerves was found to be resistant to first-line gifitinib treatment in 2020 due to a secondary EGFR T790M mutation (Fig. 2 A). After additional stereotactic body radiation therapy (SBRT), he suffered dizziness in July 2021, and MRI revealed meningeal metastasis. The patient subsequently commenced treatment with a combined regimen of savolitinib and osimertinib, resulting in a reduction in the pleural nodule, whereas the intracranial lesions remained unchanged. In July 2022, the patient exhibited cognitive decline and confusion and lower limb edema in September, with a KPS score of 40. He developed a high ICP of 200 mmHg and a high CSF-CEA level of 184 ng/mL. In addition to the previous mutations, NGS revealed novel EGFR p.S18R and EGFR p.C797S mutations in his cerebrospinal fluid as common resistance genes to osimertinib. The cytology was also positive. As a result, the patient received IP and Amivantamab beginning in October 2022. During the treatment process, a notable and substantial decrease in the CEA concentration was observed in the cerebrospinal fluid. Additionally, NGS analysis revealed a reduction in the mean mutation allele frequency (AF), which decreased from 35.84% (ranging from 0.67–95.09%) to 17.43% (ranging from 0.04–60.38%) (Fig. 2 B). Notably, cytological examination did not reveal tumor cells in the third collection of CSF samples. This resulted in a substantial alleviation of the patient's intracranial symptoms, and he continued to receive intrathecal injections of the standard dosage of pemetrexed (30 mg). In January 2023, the patient resumed treatment with osimertinib at a dose of 80 mg. As of the latest follow-up in May 2023, the patient had a progression-free survival (PFS) and overall survival (OS) of 10 months since the beginning of the IP. His mental status, appetite, sleep, and bowel movements were all reported to be normal, and his KPS score was 80. Patient 3 A 48-year-old man who had been receiving systematic treatment for more than two years was diagnosed with stage IV lung adenocarcinoma harboring a sensitive EGFR p.L858R mutation (Fig. 3 A). He was admitted to our hospital on November 18, 2022, after being diagnosed with lung cancer brain metastasis more than a year ago. In May 2020, the patient suffered from hearing loss, fatigue, dizziness, and diminished taste and appetite. Contrast-enhanced MRI of the brain revealed suspicion on leptomeningeal metastases, which was later confirmed by the presence of malignant cells in cerebrospinal fluid cytology through lumbar puncture. CSF NGS analysis revealed EGFR L858R mutation, CDK4 amplification, STK11 deletion, and MDM2 amplification, corroborating the diagnosis of lung cancer brain metastasis. In December 2020, he was administered 160 mg/day of osimertinib and anlotinib, but anlotinib was discontinued due to hematuria. The patient later experienced leg weakness that limited activity and self-care ability (KPS 40). Cytological analysis of cerebrospinal fluid obtained from a lumbar puncture revealed that the CSF was positive in March 2022, and the NGS revealed that 33.67% AF of EGFR p.L858R mutation in the CSF, indicating gradual progression of the tumor. In March 2022, the patient received 20 mg of IP. His symptoms gradually improved after the first IP treatment. Additionally, CSF NGS revealed that the AF of mutated genes was significantly lower after treatment than after initial treatment. IP was subsequently administered every 3 or 4 weeks as per the schedule. However, the CSF-ctDNA increased again, with a mean AF of 4.92% (1.61%~9.28%) at the second NGS test and 15.85% (2.79%~31.3%) at the third test (Fig. 3 B). Moreover, two months later, leg weakness recurred, and an inflexible tongue indicated the progression of an intracranial tumor. Consequently, an increased dose of pemetrexed (50 mg) was administered via intrathecal injection. At the latest follow-up, although the patient still had difficulty standing and swallowing, his condition was not very poor, and he was still alive, with an OS of as long as 14 + months since the first IP treatment. Discussion In this study, we report three patients of non-small cell lung cancer patients with leptomeningeal metastases who were treated with a combination of intrathecal pemetrexed and TKIs after the failure of TKIs alone. The treatment response was monitored using CSF ctDNA. Despite the proven efficacy of intrathecal chemotherapy, only partial patient benefit was observed. The importance of monitoring treatment response, as exemplified by patient 3 in our report, was emphasized because the patient experienced treatment responses followed by rapid relapse, which was detected through CSF ctDNA AF analysis. Given that changes in CSF ctDNA correlate with patient symptoms, monitoring ctDNA AF allows for precise dosage adjustments of pemetrexed, ensuring a tailored and effective treatment approach. Moreover, in Patient 2, no tumor cells were detected via CSF cytology after IP treatment, but ctDNA still indicated the presence of the tumor. This finding underscores the high sensitivity of NGS. The quantification of responses monitored by traditional methods is limited. For instance, imaging examination faces difficulties in depicting quantifiable size changes in dispersed leptomeningeal lesions. CSF cytological examination commonly defines tumor cells from positive specimens without quantification. In our study, we monitored and demonstrated that dynamic changes in ctDNA AF were associated with clinical treatment response; two patients experienced significant clinical relief observed through a reduction in ctDNA AF, while one patient showed a rebound in ctDNA AF. In summary, the dynamic changes in ctDNA reflect the intracranial lesion response to IP treatment and support the results obtained from imaging, cytological, and clinical performance examinations. Plasma ctDNA has demonstrated clinical applications in providing prognostic guidance through its dynamic changes (B et al. 2021, Guo et al. 2021 , AA et al. 2017 ), as well as treatment guidance, particularly medication guidance(Mok et al. 2017 , Yang et al. 2018 ), by understanding its mutations. However, there is limited prospective research on the clinical use of CSF ctDNA. Li et al. conducted a study involving 92 NSCLC patients with brain metastases and found that a ≥ 50% CSF ctDNA reduction could better predict the intracranial tumor response, leading to significantly longer intracranial progression-free survival(Li and Chen 2022). Additionally, Zheng et al. retrospectively identified the potential of CSF ctDNA in guiding targeted therapy for osimertinib-resistant leptomeningeal metastasis (LM) patients; among 22 patients who received subsequent targeted therapy based on CSF ctDNA analysis, a neurological response rate of 72.7% was achieved(Zheng et al. 2022 ). These cases provide prospective evidence supporting the practical utility of monitoring dynamic changes in CSF ctDNA during IP treatment for NSCLC leptomeningeal metastasis. However, due to the limited sample size and statistical power, an ongoing larger cohort prospective trial is currently being conducted at our hospital to further investigate the efficacy of this approach in monitoring dynamic changes in CSF ctDNA in LM patients receiving IP treatment. In summary, CSF ctDNA analysis holds promise as a reliable tool for monitoring the dynamic changes in intracranial lesions and assessing treatment responses. We present three clinical cases in which CSF ctDNA monitoring was combined with traditional methods and observed excellent consistency between changes in CSF ctDNA levels and clinical responses to IP treatment. These findings support its potential future use in routine clinical practice for LM patients. Declarations Acknowledgments We would like to thank all the patients and family members who provided their consent to present the data in this study, as well as the investigators and research staff at the hospitals and research sites involved. Ethics approval and consent to participate All individuals provided informed consent to participate in this study and approval was provided by Medical Research Ethics Committee of Guangdong Sanjiu Brain Hospital. Consent for publication Written informed consent was obtained from each patient at the time of sample submission. Author contributions Linbo Cai., Changguo Shan., Juan Li.: conceptualization, supervision, project administration; Weiping Hong., Lei Wen, Yanying Yan, Hui Wang. : methodology, data curation, investigation. Weiping Hong, Lei Wen, Chuqiao Liang, and Xiaoyu Hong: writing original draft, review and editing. Qingjun Hu, Junjie Zhen. Mingyao Lai: resources. All authors provided critical revision of the manuscript for important intellectual content. Declaration of interest C.L. and X.H. are employees of Nanjing Geneseeq Technology, Inc. All remaining authors declare no conflicts of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. References Remon J, Le Rhun E, Besse B (2017) Leptomeningeal carcinomatosis in non-small cell lung cancer patients: A continuing challenge in the personalized treatment era. Cancer Treat Rev 53:128–137 Liao BC, Lee JH, Lin CC, Chen YF, Chang CH, Ho CC et al (2015) Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors for Non-Small-Cell Lung Cancer Patients with Leptomeningeal Carcinomatosis. J Thorac oncology: official publication Int Association Study Lung Cancer 10(12):1754–1761 Umemura S, Tsubouchi K, Yoshioka H, Hotta K, Takigawa N, Fujiwara K et al (2012) Clinical outcome in patients with leptomeningeal metastasis from non-small cell lung cancer: Okayama Lung Cancer Study Group. 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Cancer Discov 7(12):1394–1403 Mok TS, Wu YL, Ahn MJ, Garassino MC, Kim HR, Ramalingam SS et al (2017) Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. N Engl J Med 376(7):629–640 Yang Z, Yang N, Ou Q, Xiang Y, Jiang T, Wu X et al (2018) Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients. Clin Cancer Res 24(13):3097–3107 Zheng MM, Li YS, Tu HY, Sun H, Yin K, Jiang BY et al (2022) Subsequent treatments beyond progression on osimertinib in EGFR-mutated NSCLC and leptomeningeal metastases. BMC Med 20(1):197 Additional Declarations Competing interest reported. C.L. and X.H. are employees of Nanjing Geneseeq Technology, Inc. All remaining authors declare no conflicts of interest. 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A.\u003c/strong\u003e The timeline of diagnosis and treatment. \u003cstrong\u003eB. \u003c/strong\u003eLongitudinal tracking of ctDNA in CSF, ICP and KPS as intracranial indicators in patient 1 during intrathecal pemetrexed treatment. \u003cstrong\u003eCSF\u003c/strong\u003ecerebrospinal fluid; \u003cstrong\u003eIP\u003c/strong\u003e intrathecal pemetrexed;\u003cstrong\u003e ICP\u003c/strong\u003e initial intracranial pressure; \u003cstrong\u003eKPS\u003c/strong\u003e Karnofsky performance score.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4482413/v1/3793206df6c007ea739bb5f5.png"},{"id":58153775,"identity":"fda61e01-b9cd-419e-b8fc-441bf128ef12","added_by":"auto","created_at":"2024-06-11 20:34:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2047474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagnosis and treatment process of patient 2. A.\u003c/strong\u003e The timeline of diagnosis and treatment. \u003cstrong\u003eB. \u003c/strong\u003eLongitudinal tracking of ctDNA in CSF, ICP and KPS as intracranial indicators in patient 2 during intrathecal pemetrexed treatment. \u003cstrong\u003eCSF\u003c/strong\u003ecerebrospinal fluid; \u003cstrong\u003eCEA\u003c/strong\u003ecarcino-embryonic antigen; \u003cstrong\u003eIP\u003c/strong\u003eintrathecal pemetrexed;\u003cstrong\u003e ICP\u003c/strong\u003e initial intracranial pressure; \u003cstrong\u003eKPS\u003c/strong\u003e Karnofsky performance score.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4482413/v1/aeb4bbb946e1ac6b9118a067.png"},{"id":58153193,"identity":"683923b5-7b52-4489-8489-131f32773914","added_by":"auto","created_at":"2024-06-11 20:26:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1631847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagnosis and treatment process of patient 3. A.\u003c/strong\u003e The timeline of diagnosis and treatment. \u003cstrong\u003eB. \u003c/strong\u003eLongitudinal tracking of ctDNA in CSF, ICP and KPS as intracranial indicators in patient 3 during intrathecal pemetrexed treatment. \u003cstrong\u003eCSF\u003c/strong\u003ecerebrospinal fluid; \u003cstrong\u003eIP\u003c/strong\u003e intrathecal pemetrexed;\u003cstrong\u003e ICP\u003c/strong\u003e initial intracranial pressure; \u003cstrong\u003eKPS\u003c/strong\u003e Karnofsky performance score.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4482413/v1/853cf92ae5210cb88801ee33.png"},{"id":58155422,"identity":"49a2c7cf-a9ef-4652-b5ac-feaaba80199b","added_by":"auto","created_at":"2024-06-11 20:50:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6263596,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4482413/v1/91a832d2-4c80-45d6-9586-eb669732dca9.pdf"}],"financialInterests":"Competing interest reported. C.L. and X.H. are employees of Nanjing Geneseeq Technology, Inc. All remaining authors declare no conflicts of interest.","formattedTitle":"Advancing Non-Small Cell Lung Cancer Treatment: A Case Series on Pemetrexed Intrathecal Chemotherapy Monitored Through Circulating Tumor DNA in Cerebrospinal Fluid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately 5% of patients with advanced non-small cell lung cancer (NSCLC) may develop leptomeningeal metastasis (LM), which is more prevalent in those with \u003cem\u003eEGFR\u003c/em\u003e mutations. (Remon et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Liao et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)Patients with LM and a low performance status (PS) may have a poor prognosis; the median overall survival (OS) of LM NSCLC patients is only 3.6 to 11 months(Liao and Lee 2015, Umemura et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Although targeted therapies, particularly tyrosine kinase inhibitors (TKIs), are effective against primary tumors, the blood‒brain barrier (BBB) may limit intracranial performance, resulting in variable patient survival outcomes. (Cheng et al. 2018) LM patients have been shown to benefit from a variety of treatments, including surgery, radiation, systematic chemotherapy, targeted chemotherapy, immunotherapy, and intrathecal injection, but there is still a lack of conventional treatment paradigms for LM patients. In patients with NSCLC, intrathecal administration, which delivers medications directly into the subarachnoid space via cerebrospinal fluid (CSF), was reported to be highly effective in controlling LM. (Roguski et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Pan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) A recent clinical trial has proven the efficacy and safety of intrathecal pemetrexed combined with dexamethasone for treating TKI-failed LM EGFR\u0026thinsp;+\u0026thinsp;NSCLC. At the same time, they revealed the challenge of their study on measuring treatment responses.(Fan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eEvaluating the efficacy of treatment for LM remains difficult and lacks standardization. Due to the diffusion of LM lesions, it is challenging to quantify tumor size using imaging techniques. The Karnofsky performance score (KPS) is a commonly used tool for assessing functional impairment in LM patients (Schag et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), but it assesses treatment efficacy indirectly through patient performance and cannot reveal tumor changes in a quantifiable manner. Response Assessment in Neuro-Oncology (RANO) is a generally accepted response criterion that evaluates treatment response by incorporating a novel radiographic scorecard, CSF cytological examination or flow cytometry, and neurological examination, but it is limited in its ability to measure lesions for response assessment. (Chamberlain et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Le Rhun et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) On the other hand, the significant issue of high false negatives in the detection of cerebrospinal fluid cytology also poses a major challenge in monitoring treatment effectiveness.(Hyun et al. 2016) Recent research has demonstrated that circulating tumor DNA (ctDNA) released by tumor apoptosis can be used as a biomarker to track treatment responses and tumor evolution. (Diehl et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) CtDNA from cerebrospinal fluid (CSF) has also demonstrated superior intracranial response prediction performance compared to that from plasma. (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe urgent need for standardization of intrathecal chemotherapy, a novel and effective treatment for patients with LM, is inextricably linked to the establishment of quantifiable evaluation methods. In this study, we employed Next-generation sequencing (NGS) to identify ctDNA in CSF from three patients diagnosed with LM. These patients received intrathecal pemetrexed (IP) therapy, which was administered as an initial single dose of 20 mg in week 1, followed by 30 mg in week 3. Subsequently, the patients continued to receive doses of 30 mg to 50 mg once every three weeks, as indicated by changes in the NGS allele frequency (AF) until disease progression or intolerance occurred. To the best of our knowledge, this is the first report on therapeutic responses and clinical observations of this approach.\u003c/p\u003e"},{"header":"Patient 1","content":"\u003cp\u003eIn October 2022, a 66-year-old female patient who had been treated with surgery and EGFR-TKIs for lung adenocarcinoma metastases since 2016 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) visited our hospital due to numbness in her left hand. Her mental state, appetite, and sleep were all normal, yet she was experiencing numbness in her limbs, particularly in her left hand, which significantly affected her ability to perform daily self-care tasks (KPS 50). In addition to positive cytology of the CFS, MRI revealed a new lesion on her meninges, leading to a diagnosis of lung adenocarcinoma meningeal metastases. After the failure of targeted treatments, between October 2022 and March 2023, the patient underwent six IP treatments, with an initial intracranial pressure (ICP) of 210 mmHg for the first time. The collection of ctDNA from her CSF began prior to receiving the third IP. Along with significant symptomatic improvement, we observed a reduction in the mutation allele frequency (AF) of ctDNA in both the cerebrospinal fluid (CSF) and intracranial pressure (ICP) throughout the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Specifically, the mean AF decreased from 51.94% (ranging from 19.42\u0026ndash;90.49%) to 19.39% (ranging from 7.97\u0026ndash;43.22%). Her capacity to walk, engage in verbal communication, and maneuver her limbs was enhanced, allowing her to regain her self-care abilities (KPS 80). A general dose (30 mg) of pemetrexed was given, as both NGS and clinical response improved. Retrospectively, the \u003cem\u003eEGFR\u003c/em\u003e L858R mutation was identified by NGS testing after surgical treatment of metastatic front parietal intra-axial brain tumors in 2016. She was treated with gefitinib for more than two years and then switched to osimertinib in 2019 due to the slow progression of her brain metastases. Gamma-knife surgery was performed for brain tumors in 2020. Although the patient had been taking osimertinib for twenty-four months without experiencing headache or dizziness, limb numbness caused by meningeal metastases did not improve. The local IP treatment relieved numbness in her limbs, which was observed through the decrease in ctDNA AF in CSF, along with improvements in KPS and ICP. From the start of the intrathecal pemetrexed treatment until the last follow-up in May 2023, the patient had a progression-free survival (PFS) of 6 months and an overall survival (OS) of 6 months.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Patient 2","content":"\u003cp\u003eA 66-year-old male patient who had a 30-year history of smoking, diagnosed with moderately differentiated adenocarcinoma with metastases in the lungs, lymph nodes, bones and nerves was found to be resistant to first-line gifitinib treatment in 2020 due to a secondary \u003cem\u003eEGFR\u003c/em\u003e T790M mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After additional stereotactic body radiation therapy (SBRT), he suffered dizziness in July 2021, and MRI revealed meningeal metastasis. The patient subsequently commenced treatment with a combined regimen of savolitinib and osimertinib, resulting in a reduction in the pleural nodule, whereas the intracranial lesions remained unchanged. In July 2022, the patient exhibited cognitive decline and confusion and lower limb edema in September, with a KPS score of 40. He developed a high ICP of 200 mmHg and a high CSF-CEA level of 184 ng/mL. In addition to the previous mutations, NGS revealed novel \u003cem\u003eEGFR\u003c/em\u003e p.S18R and \u003cem\u003eEGFR\u003c/em\u003e p.C797S mutations in his cerebrospinal fluid as common resistance genes to osimertinib. The cytology was also positive. As a result, the patient received IP and Amivantamab beginning in October 2022. During the treatment process, a notable and substantial decrease in the CEA concentration was observed in the cerebrospinal fluid. Additionally, NGS analysis revealed a reduction in the mean mutation allele frequency (AF), which decreased from 35.84% (ranging from 0.67–95.09%) to 17.43% (ranging from 0.04–60.38%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, cytological examination did not reveal tumor cells in the third collection of CSF samples. This resulted in a substantial alleviation of the patient's intracranial symptoms, and he continued to receive intrathecal injections of the standard dosage of pemetrexed (30 mg). In January 2023, the patient resumed treatment with osimertinib at a dose of 80 mg. As of the latest follow-up in May 2023, the patient had a progression-free survival (PFS) and overall survival (OS) of 10 months since the beginning of the IP. His mental status, appetite, sleep, and bowel movements were all reported to be normal, and his KPS score was 80.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Patient 3","content":"\u003cp\u003eA 48-year-old man who had been receiving systematic treatment for more than two years was diagnosed with stage IV lung adenocarcinoma harboring a sensitive \u003cem\u003eEGFR\u003c/em\u003e p.L858R mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). He was admitted to our hospital on November 18, 2022, after being diagnosed with lung cancer brain metastasis more than a year ago. In May 2020, the patient suffered from hearing loss, fatigue, dizziness, and diminished taste and appetite. Contrast-enhanced MRI of the brain revealed suspicion on leptomeningeal metastases, which was later confirmed by the presence of malignant cells in cerebrospinal fluid cytology through lumbar puncture. CSF NGS analysis revealed \u003cem\u003eEGFR\u003c/em\u003e L858R mutation, \u003cem\u003eCDK4\u003c/em\u003e amplification, \u003cem\u003eSTK11\u003c/em\u003e deletion, and \u003cem\u003eMDM2\u003c/em\u003e amplification, corroborating the diagnosis of lung cancer brain metastasis. In December 2020, he was administered 160 mg/day of osimertinib and anlotinib, but anlotinib was discontinued due to hematuria. The patient later experienced leg weakness that limited activity and self-care ability (KPS 40). Cytological analysis of cerebrospinal fluid obtained from a lumbar puncture revealed that the CSF was positive in March 2022, and the NGS revealed that 33.67% AF of \u003cem\u003eEGFR\u003c/em\u003e p.L858R mutation in the CSF, indicating gradual progression of the tumor. In March 2022, the patient received 20 mg of IP. His symptoms gradually improved after the first IP treatment. Additionally, CSF NGS revealed that the AF of mutated genes was significantly lower after treatment than after initial treatment. IP was subsequently administered every 3 or 4 weeks as per the schedule. However, the CSF-ctDNA increased again, with a mean AF of 4.92% (1.61%~9.28%) at the second NGS test and 15.85% (2.79%~31.3%) at the third test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, two months later, leg weakness recurred, and an inflexible tongue indicated the progression of an intracranial tumor. Consequently, an increased dose of pemetrexed (50 mg) was administered via intrathecal injection. At the latest follow-up, although the patient still had difficulty standing and swallowing, his condition was not very poor, and he was still alive, with an OS of as long as 14 + months since the first IP treatment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we report three patients of non-small cell lung cancer patients with leptomeningeal metastases who were treated with a combination of intrathecal pemetrexed and TKIs after the failure of TKIs alone. The treatment response was monitored using CSF ctDNA. Despite the proven efficacy of intrathecal chemotherapy, only partial patient benefit was observed. The importance of monitoring treatment response, as exemplified by patient 3 in our report, was emphasized because the patient experienced treatment responses followed by rapid relapse, which was detected through CSF ctDNA AF analysis. Given that changes in CSF ctDNA correlate with patient symptoms, monitoring ctDNA AF allows for precise dosage adjustments of pemetrexed, ensuring a tailored and effective treatment approach. Moreover, in Patient 2, no tumor cells were detected via CSF cytology after IP treatment, but ctDNA still indicated the presence of the tumor. This finding underscores the high sensitivity of NGS.\u003c/p\u003e \u003cp\u003eThe quantification of responses monitored by traditional methods is limited. For instance, imaging examination faces difficulties in depicting quantifiable size changes in dispersed leptomeningeal lesions. CSF cytological examination commonly defines tumor cells from positive specimens without quantification. In our study, we monitored and demonstrated that dynamic changes in ctDNA AF were associated with clinical treatment response; two patients experienced significant clinical relief observed through a reduction in ctDNA AF, while one patient showed a rebound in ctDNA AF. In summary, the dynamic changes in ctDNA reflect the intracranial lesion response to IP treatment and support the results obtained from imaging, cytological, and clinical performance examinations.\u003c/p\u003e \u003cp\u003ePlasma ctDNA has demonstrated clinical applications in providing prognostic guidance through its dynamic changes (B et al. 2021, Guo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, AA et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as well as treatment guidance, particularly medication guidance(Mok et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), by understanding its mutations. However, there is limited prospective research on the clinical use of CSF ctDNA. Li et al. conducted a study involving 92 NSCLC patients with brain metastases and found that a\u0026thinsp;\u0026ge;\u0026thinsp;50% CSF ctDNA reduction could better predict the intracranial tumor response, leading to significantly longer intracranial progression-free survival(Li and Chen 2022). Additionally, Zheng et al. retrospectively identified the potential of CSF ctDNA in guiding targeted therapy for osimertinib-resistant leptomeningeal metastasis (LM) patients; among 22 patients who received subsequent targeted therapy based on CSF ctDNA analysis, a neurological response rate of 72.7% was achieved(Zheng et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese cases provide prospective evidence supporting the practical utility of monitoring dynamic changes in CSF ctDNA during IP treatment for NSCLC leptomeningeal metastasis. However, due to the limited sample size and statistical power, an ongoing larger cohort prospective trial is currently being conducted at our hospital to further investigate the efficacy of this approach in monitoring dynamic changes in CSF ctDNA in LM patients receiving IP treatment.\u003c/p\u003e \u003cp\u003eIn summary, CSF ctDNA analysis holds promise as a reliable tool for monitoring the dynamic changes in intracranial lesions and assessing treatment responses. We present three clinical cases in which CSF ctDNA monitoring was combined with traditional methods and observed excellent consistency between changes in CSF ctDNA levels and clinical responses to IP treatment. These findings support its potential future use in routine clinical practice for LM patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the patients and family members who provided their consent to present the data in this study, as well as the investigators and research staff at the hospitals and research sites involved.\u003c/p\u003e\n\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll individuals provided informed consent to participate in this study and approval was provided by Medical Research Ethics Committee of Guangdong Sanjiu Brain Hospital.\u003c/p\u003e\n\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from each patient at the time of sample submission.\u003c/p\u003e\n\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eLinbo Cai., Changguo Shan., Juan Li.: conceptualization, supervision, project administration; Weiping Hong., Lei Wen, Yanying Yan, Hui Wang. : methodology, data curation, investigation. Weiping Hong, Lei Wen, Chuqiao Liang, and Xiaoyu Hong: writing original draft, review and editing. Qingjun Hu, Junjie Zhen. Mingyao Lai: resources. All authors provided critical revision of the manuscript for important intellectual content.\u003c/p\u003e\n\n\u003cp\u003eDeclaration of interest\u003c/p\u003e\n\u003cp\u003eC.L. and X.H. are employees of Nanjing Geneseeq Technology, Inc. All remaining authors declare no conflicts of interest.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRemon J, Le Rhun E, Besse B (2017) Leptomeningeal carcinomatosis in non-small cell lung cancer patients: A continuing challenge in the personalized treatment era. Cancer Treat Rev 53:128\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao BC, Lee JH, Lin CC, Chen YF, Chang CH, Ho CC et al (2015) Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors for Non-Small-Cell Lung Cancer Patients with Leptomeningeal Carcinomatosis. J Thorac oncology: official publication Int Association Study Lung Cancer 10(12):1754\u0026ndash;1761\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmemura S, Tsubouchi K, Yoshioka H, Hotta K, Takigawa N, Fujiwara K et al (2012) Clinical outcome in patients with leptomeningeal metastasis from non-small cell lung cancer: Okayama Lung Cancer Study Group. Lung cancer (Amsterdam. Netherlands) 77(1):134\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng H, Perez-Soler R (2018) Leptomeningeal metastases in non-small-cell lung cancer. 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Clin Cancer Res 24(13):3097\u0026ndash;3107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng MM, Li YS, Tu HY, Sun H, Yin K, Jiang BY et al (2022) Subsequent treatments beyond progression on osimertinib in EGFR-mutated NSCLC and leptomeningeal metastases. BMC Med 20(1):197\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4482413/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4482413/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntrathecal pemetrexed (IP) treatment represents a promising approach for managing leptomeningeal metastasis (LM) in cancer patients. However, a standardized and measurable method to evaluate the efficacy of IP for non-small cell lung cancer (NSCLC) patients with LM is currently lacking. This report describes three NSCLC-LM cases treated with IP following progression with tyrosine kinase inhibitors (TKIs) alone. We observed their responses through next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF). The outcomes were favorable for patient 1 and 2, whereas patient 3 experienced a relapse. Notably, changes in the allele frequency (AF) of ctDNA mutations corresponded with clinical outcomes across these patients, which were also corroborated by multiple traditional clinical markers. This observation is significant, particularly in patient 3, where ctDNA monitoring effectively described the patient's temporary improvement followed by deterioration. Furthermore, in patient 2, the transition from a positive to a negative cytological test, alongside persistent positive NGS results, underscores the higher sensitivity of NGS compared to conventional cytological analysis. This suggests that longitudinal ctDNA monitoring using CSF samples may serve as an effective and independent method for assessing and dynamically tracking the response to IP treatment in NSCLC-LM patients. This approach has the potential to refine therapeutic strategies and improve patient outcomes.\u003c/p\u003e","manuscriptTitle":"Advancing Non-Small Cell Lung Cancer Treatment: A Case Series on Pemetrexed Intrathecal Chemotherapy Monitored Through Circulating Tumor DNA in Cerebrospinal Fluid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 20:26:13","doi":"10.21203/rs.3.rs-4482413/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe073b02-c85f-4304-a98a-f444cd61fad7","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-11T20:26:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-11 20:26:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4482413","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4482413","identity":"rs-4482413","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}