Cerebrospinal Fluid Circulating Tumor Cells Research Articles

BIOM-58. RE-INDUCTION OF INTRATHECAL CHEMOTHERAPY IN PRIOR RESPONDERS AND CORRELATION WITH CEREBROSPINAL FLUID CIRCULATING TUMOR CELLS

Abstract INTRODUCTION Leptomeningeal carcinomatosis (LMC) is an aggressive form of metastasis, with a historically poor median overall survival. Newer therapies and treatment strategies are improving outcomes, and more long-term responders exist. However, there are limited options for intrathecal (IT) chemotherapy, and re-induction in prior responders may be a reasonable treatment strategy. In addition, quantitative cerebrospinal fluid circulating tumor cells (CSF CTCs) are a valuable tool in LMC, for both surveillance of recurrence and evaluation of response. METHODS We retrospectively identified 10 patients with LMC who showed a response to IT chemotherapy and were followed with quantitative CSF CTCs on a commercially available platform. At progression, patients underwent re-induction of IT chemotherapy, on a twice a week schedule, and were evaluated for response. RESULTS All 10 patients were treated with re-induction IT chemotherapy. Six patients were re-challenged with topotecan, 3 with topotecan + trastuzumab, and 1 with pemetrexed + trastuzumab. Six patients had a primary diagnosis of breast cancer, three with lung cancer and one with ovarian cancer. The median KPS was 70. Eight were female. Prior to re-induction, all 10 had an increase in quantitative CSF CTCs. Only 1/10 had new symptoms, and 5/10 showed imaging changes on MRI. At the time of re-induction, 3/10 also received stereotactic radiosurgery to brain metastases and 2/10 had a change in systemic chemotherapy. 9/10 had improved CSF CTC counts on the next restaging, typically at 8 weeks. The median overall survival from re-challenge was 12.7 months. CONCLUSION Quantitative CSF CTCs were a more sensitive predictor of recurrence than symptoms or imaging, and were a marker of response to re-induction IT chemotherapy. Re-induction showed a durable survival benefit, and is a reasonable treatment strategy in patients who were prior responders

Applications of cerebrospinal fluid circulating tumor cells and circulating tumor-derived DNA in diagnosis, prognosis, and personalized treatment of CNS metastases.

A common feature of advanced solid tumors is their ability to metastasize and colonize distant organs, including the Central Nervous System (CNS), which encompasses brain and leptomeningeal metastases (LM). While cerebrospinal fluid cytopathological analysis remains a gold standard diagnostic tool, it only provides limited insights into the biology of tumor cells; thus, it is urgent to develop minimally invasive biomarkers that enable a comprehensive quantitative and molecular characterization of disseminated cells, therapy response assessment, and disease monitoring. Liquid biopsy methods have been swiftly developed for some readily accessible bodily fluids such as plasma and urine; circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from these sources have been rapidly implemented into clinical trial design, disease monitoring, and treatment assignment across different tumor types. However, the filter imposed by the brain blood barrier (BBB) hampers the release of tumor-derived cells and molecules from CNS metastases. Crucially, cerebrospinal fluid (CSF) liquid biopsy methods offer a unique and unparallel source to develop liquid biopsy methodologies in patients with CNS-disseminated disease, including the characterization of CTCs and ctDNA arising specifically from brain and leptomeningeal metastasis. These technologies have enabled a deeper understanding of tumor cell and molecular dynamics, including the reconstruction of clonal evolution in the brain microenvironment through longitudinal sapling. Here, we discuss the current challenges and opportunities that CSF liquid biopsy methods face for the implementation of these approaches into clinical settings.

Clinical features and outcomes of patients with breast cancer with leptomeningeal carcinomatosis.

e13162 Background: Leptomeningeal carcinomatosis (LMC) is a rare, but deadly complication in breast cancer, with prior studies showing median overall survival (OS) of 15 weeks. There is a paucity of data on histologic and receptor subtype-specific survival, and there is no standard of care currently for treatment of LMC in breast cancer. We reviewed the outcomes of a contemporary cohort of breast cancer patients (pts) with LMC. Methods: This is a single-institution retrospective analysis of 38 female breast cancer pts diagnosed with LMC between 2016-2023, based on radiographic features and/or positive cerebrospinal fluid (CSF) cytology or CSF circulating tumor cells (CTCs). Patients consented to share information with a research biorepository, and we conducted a chart review of their clinical features and OS with LMC. Results: 25 pts (65.8%) had invasive ductal carcinoma (IDC), 7 pts (18.4%) had invasive lobular carcinoma (ILC), 4 (10.5%) had metaplastic carcinoma, and 2 (5.3%) had mixed IDC/ILC. 22 pts (57.9%) had triple-negative breast cancer (TNBC), 12 (31.6%) had estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-), and 4 (10.5%) had HER2+ disease. Median age at LMC diagnosis was 50 years. Median time between Stage IV and LMC diagnosis was 11 months. Pts received a median of 2 lines of systemic therapy for metastatic disease prior to LMC diagnosis. 11 pts (28.9%) had parenchymal brain metastasis prior to LMC diagnosis. 32 pts (84.2%) had signs of LMC on Magnetic Resonance Imaging of the brain and/or spine at diagnosis. 25 pts underwent CSF cytology and 13 pts underwent CSF CTC evaluation at LMC diagnosis; 14/25 pts (56%) had positive CSF cytology and 10/13 pts (76.9%) had detectable CSF CTCs. 21 pts (55.3%) underwent whole-brain radiotherapy, and 2 pts (5.3%) received proton craniospinal irradiation. 21 pts (55.3%) received IT therapy. 27 pts (71.1%) received at least 1 line of systemic therapy post-LMC diagnosis. Median OS between LMC diagnosis and death or last oncology follow up for the entire cohort was 5 months (range 0-57 months, 95% CI 3.0-8.0). There was a significant difference in median OS with LMC by receptor subtype: TNBC 3.5 months, ER+/HER2- 8 months, and HER2+ 23 months (p=0.0047). Two HER2+ pts survived ≥ 3 years post-LMC diagnosis with anti-HER2 antibody-drug conjugates and tyrosine kinase inhibitors +/- stereotactic radiosurgery (SRS) to parenchymal brain metastases. Amongst 9 pts with ER+/HER2-low disease, longer survival with LMC (≥ 16 months) was observed with trastuzumab deruxtecan, IT topotecan +/- IT trastuzumab, and SRS to parenchymal brain metastases. Conclusions: This study highlights heterogeneity in outcomes amongst breast cancer pts with LMC by receptor subtype, as well as improvement in outcomes for selected pts treated with novel therapeutics and a multidisciplinary approach. Larger prospective studies are needed to inform treatment paradigms for LMC.

Real-world use of a CSF circulating tumor cell assay in the diagnosis and management of leptomeningeal metastasis.

2029 Background: The diagnosis of leptomeningeal metastasis (LM) can be challenging due to variability in clinical symptoms, radiographic, and cerebrospinal fluid (CSF) findings. The National Comprehensive Cancer Network (NCCN) guidelines recommend assessment of circulating tumor DNA (ctDNA) to increase sensitivity of tumor cell detection and assess the response to treatment. We hypothesize that the real-world use of an assay for the direct detection and enumeration of circulating tumor cells (CTCs) in the CSF is clinically useful for accurate diagnosis of LM, particularly in an early stage of disease. Methods: We report a series of 55 patients treated at our institution with a cancer diagnosis and either neurological symptoms or radiographic findings that suggested LM. All patients underwent a lumbar puncture, assessment for CSF CTCs, and conventional hospital-based cytology testing between 6/1/2020 and 8/1/2023. Survival data cutoff was 12/31/2023. Patient outcomes are reported. Results: 55 patients were tested, and 30 patients were found to be positive for CTCs and diagnosed with LM. Only 10 patients were concurrently positive for CTCs and cytology at our institution, whereas 20 patients with positive CTCs had a negative or ambiguous cytology. No patients with a negative CTC result (25 patients) were subsequently diagnosed with LM, whereas 4 patients with a positive CTC result remained without progressive neurological symptoms. 5 patients diagnosed with LM were alive at the time of data cutoff. The histologic breakdown for LM was: breast (11), non-small cell lung cancer (NSCLC) (16), gastrointestinal (GI) (3). Median overall survival (OS) was longer in patients with a positive CTC and negative cytology result compared to patients with concurrent positive CTC and cytology (172 vs. 63 days, p = 0.06). Median OS was also longer in breast cancer LM compared to NSCLC LM (235 vs. 63 days, p = 0.008). Most patients diagnosed with LM received therapies including Ommaya reservoir placement, intrathecal chemotherapy, and/or radiation. Conclusions: The use of a CSF circulating tumor cell assay aided the diagnosis of LM and led to timely initiation of treatment. A negative result of the CTC assay was associated with ruling out LM. Diagnosis of LM with the CTC assay and negative cytology was associated with longer survival, possibly due to earlier treatment initiation, and not solely a lead time bias. Breast cancer LM patients had improved survival compared to NSCLC and GI cancer patients, possibly due to more effective treatment options.

SDPS-47 CIRCULATING TUMOR CELL ANALYSIS FROM THE CEREBROSPINAL FLUID INFORMS EARLY DIAGNOSIS, TREATMENT AND PROGNOSIS IN LEPTOMENINGEAL CARCINOMATOSIS

Abstract INTRODUCTION Leptomeningeal carcinomatosis (LMC) is an aggressive pattern of recurrence with limited survival. Early detection by traditional CSF (cerebrospinal fluid) cytology is poor. We evaluated CSF circulating tumor cells (CTCs) with a commercially available assay (CNSideTM) and subsequent molecular profiling on captured CTCs, to determine if CTC analysis would lead to early detection, identification of therapeutic targets, and improved treatment outcomes. METHODS We analyzed 58 patients with carcinoma who were referred to Neuro-oncology clinic for evaluation for LMC, and underwent comprehensive work-up with CSF sampling (traditional cytology and CTCs), brain and spine MRI, and neurologic examination, and were followed for survival. RESULTS 28/58 were diagnosed with LMC (breast n=16, Lung n=9, other n=3). 24/28 were female. Median KPS was 80. 15/16 breast cancer patients were HER2- on initial systemic cancer diagnosis. The mean time to LMC from cancer diagnosis was 70 months. In the patients with LMC, CTCs were positive in 96%, compared to traditional cytology, which was positive in 64%. Molecular analysis of the CTCs in 15 patients with HER2- primary tumors detected HER2+ amplification in 3/15 patients. 14/28 patients received first line therapy for LMC with radiation + systemic chemotherapy + intrathecal chemotherapy, and 8/28 received systemic chemotherapy + intrathecal chemotherapy. 6/28 received other combinations. The overall median progression-free survival (PFS) was 5.3 months and the overall survival (OS) was 7.7 months from LMC diagnosis (breast PFS 4.5 months, OS 6.3 months; Lung PFS 5.3 months, OS 23.5 months). CONCLUSION CTC detection had superior sensitivity compared to traditional cytology, allowing for earlier detection of LMC and consequently earlier treatment. Molecular analysis of CTCs can allow for identification of therapeutic targets specific to the CSF, such as intrathecal trastuzumab for HER2+ LMC. PFS and OS in our cohort with this approach suggested improved survival compared to historical controls.

A phase Ia/Ib study of intrathecal deferoxamine in patients with leptomeningeal metastases.

TPS2074 Background: Leptomeningeal metastases (LM) represent an aggressive form of advanced cancer with few durable therapeutic options. One of the principal barriers in treating LM is the paucity of knowledge on cancer cell survival and proliferation within the nutrient-sparse cerebrospinal fluid (CSF). Single-cell RNA sequencing of patient-derived CSF has identified that cancer cells in the spinal fluid employ the single iron-binding transporter and receptor system, lipocalin-2/SLC22A17, to gather sparse iron to sustain their metabolic needs. This phenotype is recapitulated in preclinical mouse models of LM. Depletion of CSF iron via intracisternal administration of deferoxamine, a parenteral iron chelator, dramatically reduced LM growth and significantly prolonged survival in preclinical models. Exploiting LM iron dependency using intrathecal deferoxamine (IT-DFO) represents a novel therapeutic approach for patients with LM. Methods: This is a prospective, open-label, single center phase Ia dose escalation study of IT-DFO in patients with LM from any solid tumor malignancy to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D), followed by a phase Ib dose expansion of IT-DFO at the RP2D in patients with LM from non-small-cell lung cancer (NSCLC). Eligibility criteria include newly diagnosed or recurrent LM identified by magnetic resonance imaging (MRI), positive CSF cytology, and/or elevated CSF circulating tumor cells (CTCs), age ≥ 18 years, Karnofsky Performance Status ≥ 60, and life expectancy ≥ 8 weeks. All patients will receive IT-DFO in 28-day cycles at a frequency of twice weekly (cycle 1), once weekly (cycle 2), and once every two weeks (cycle 3+). Patients will be monitored for LM progression by neurological examination, neuraxial MRI, and CSF cytology as per modified Response Assessment in Neuro-Oncology LM criteria. Phase Ia will involve a modified accelerated titration over 9 dosing cohorts (IT-DFO dose range 10mg to 495mg) with monitoring for dose-limiting toxicities until the MTD is reached. Phase Ib will further explore the safety of IT-DFO at the RP2D in 20 patients with NSCLC LM. Secondary objectives include the determination of pharmacokinetic and pharmacodynamic properties of IT-DFO and its metabolite, ferrioxamine, in the CSF and serum (phase Ia/Ib), and efficacy outcomes (phase Ib) including LM-objective response rate, LM-clinical benefit rate, LM-duration of response, LM-progression-free survival, and overall survival. Exploratory analyses will prospectively correlate CSF CTC enumeration with treatment response and characterize the impact of IT-DFO on cancer cell metabolism, resistance pathways, and the CSF immune microenvironment. Clinical trial information: NCT05184816.

A preclinical model of patient-derived cerebrospinal fluid circulating tumor cells for experimental therapeutics in leptomeningeal disease from melanoma.

BackgroundLeptomeningeal disease (LMD) occurs as a late complication of several human cancers and has no rationally designed treatment options. A major barrier to developing effective therapies for LMD is the lack of cell-based or preclinical models that recapitulate human disease. Here, we describe the development of in vitro and in vivo cultures of patient-derived cerebrospinal fluid circulating tumor cells (PD-CSF-CTCs) from patients with melanoma as a preclinical model to identify exploitable vulnerabilities in melanoma LMD.MethodsCSF-CTCs were collected from melanoma patients with melanoma-derived LMD and cultured ex vivo using human meningeal cell-conditioned media. Using immunoassays and RNA-sequencing analyses of PD-CSF-CTCs, molecular signaling pathways were examined and new therapeutic targets were tested for efficacy in PD-CSF-CTCs preclinical models.ResultsPD-CSF-CTCs were successfully established both in vitro and in vivo. Global RNA analyses of PD-CSF-CTCs revealed several therapeutically tractable targets. These studies complimented our prior proteomic studies highlighting IGF1 signaling as a potential target in LMD. As a proof of concept, combining treatment of ceritinib and trametinib in vitro and in vivo demonstrated synergistic antitumor activity in PD-CSF-CTCs and BRAF inhibitor-resistant melanoma cells.ConclusionsThis study demonstrates that CSF-CTCs can be grown in vitro and in vivo from some melanoma patients with LMD and used as preclinical models. These models retained melanoma expression patterns and had signaling pathways that are therapeutically targetable. These novel models/reagents may be useful in developing rationally designed treatments for LMD.

Long-Term Outcomes of a Prospective Clinical Trial of Proton Craniospinal Irradiation for Leptomeningeal Metastasis

Leptomeningeal metastasis (LM) is associated with limited survival and treatment options for patients with solid tumor malignancies. While photon involved-field radiotherapy (IFRT) is effective for local palliation, it lacks durability. Craniospinal irradiation (CSI), in contrast, treats the entire central nervous system (CNS) compartment, thus potentially improving disease control. We previously reported the safety and tolerability of proton CSI (pCSI) in a completed phase IB study, and we are now reporting the mature survival outcomes of these patients.We enrolled 24 patients with solid tumor LM to receive hypofractionated pCSI of 30 Gy (RBE) in 10 fractions in this phase I prospective trial between June 2018 and May 2019 (NCT03520504). The primary endpoint is dose-liming toxicity (DLT) within 1 month of treatment. Secondary endpoints, including time to CNS progression (CNS TTP), CNS progression-free survival (PFS), and overall survival (OS), were assessed using Kaplan-Meier analysis with median survival estimates and 95% confidence intervals (CI) reported. Correlations between cerebrospinal fluid (CSF) circulating tumor cells (CTCs) and survival outcomes were evaluated using Kaplan-Meier analysis. An optimal cutoff for CTCs was determined through maximally selected rank statistics using CNS PFS.Of the 24 patients enrolled, 3 did not complete treatment due to systemic disease progression and were excluded from this analysis. The majority of patients had metastatic lung (52%) and breast (33%) malignancies. The median age was 52 years (range 30-67 years) and the median Karnofsky performance status was 70 (range 60-90). The majority of patients had LM that progressed on other therapies prior to enrollment (57% vs. 43% of patients with newly diagnosed LM), and all patients with molecularly driven disease had CNS progression on small-molecule tyrosine kinase inhibitors prior to enrollment. Only 1 patient was censored at 24 months of follow-up, while 20 patients were followed until death with a median OS of 9 months (95% CI 6-22 months). The median CNS TTP was 8 months (95% CI 7-18 months), with 5 (24%) patients with durable CNS control for > 12 months after pCSI. The median CNS PFS was 7 months (95% CI 5-13 months). In the 18 patients with CSF CTCs evaluation at baseline, having a pre-pCSI CSF CTCs < 41/3mL was associated with improved CNS TTP (19 vs. 8 months, P < 0.01), CNS PFS (18 vs. 6 months, P < 0.01), and a trend toward improved OS (21 vs. 8 months, P = 0.06).We reported that pCSI is a safe treatment for patients with solid tumor LM. In this analysis, we demonstrated durable disease control and prolonged survival in some patients. Lower CSF CTCs at baseline, a potential biomarker of response, was associated with improved survival outcomes. An ongoing randomized phase II trial will determine the efficacy of pCSI compared to photon IFRT in patients with solid tumor LM.

BIOL-04. CYTOPLASM PROTEIN GFAP MAGNETIC BEADS CONSTRUCTION AND APPLICATION AS CELL SEPARATION TARGET FOR BRAIN TUMORS

BackgroundIt is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice. In contrast to the past use of epithelial EpCAM as CTCs separation target, a cytoplasm protein of GFAP antibody was first selected to construct highly-sensitive immunomagnetic liposome beads (IMLs). The validation and efficiency of this system in capturing CTCs for brain tumors were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed. ResultsOur data show that CTCs can be successfully isolated from CSF and blood samples from 32 children with brain tumors. The numbers of CTCs in CSF were significantly higher than those in blood. The level of CTCs in CSF was related to the type and location of the tumor rather than its stage. The higher the CTCs number is, the more possibly the patient will suffer from poor prognosis. Genetic testing in GFAP CTC-DNA by sanger sequencing, q-PCR and NGS methods indicated that the isolated CTCs (GFAP+/EGFR+) are the related tumor cell. For example, the high expression of NPR3 gene in CSF CTCs was consistent with that of tumor tissue. ConclusionsThe results indicated that GFAP-IML CTCs isolation system, combined with an EGFR immunofluorescence assay of antitumor marker, can serve as a brand-new method for the identification of CTCs for brain tumors. Via lumbar puncture, a minimally invasive procedure, this technique may play a significant role in the clinical diagnosis and drug evaluation of brain tumors.

Cerebrospinal fluid circulating tumor cells as a predictive biomarker for proton craniospinal irradiation for leptomeningeal metastases.

2011 Background: Leptomeningeal metastasis (LM) involves seeding of tumor cells to the cerebrospinal fluid (CSF) and the leptomeninges. Proton craniospinal irradiation (pCSI) has been shown to be potentially effective for patients with solid tumor LM. We evaluated whether CSF circulating tumor cells (CSF-CTC) and neuroimaging correlate with outcomes in patients with LM treated with pCSI. Methods: We reviewed a single-institution retrospective database of patients treated with pCSI for LM between 2018-2020 who had ≥ 3 months (mos.) follow-up and identified 58 patients. Pre-pCSI CSF-CTC using CellSearch and magnetic resonance imaging (MRI) data, and post-pCSI CSF-CTC nadir before initiation of new cancer-directed therapy were assessed. The optimal cutoff for pre-pCSI CSF-CTC was determined using maximally selected rank statistics. Kaplan Meier analysis was used to identify univariate correlates with CNS progression free survival (CNS PFS) and overall survival (OS), calculated from start of pCSI. Multivariate Cox proportional hazards modeling was used to test independence of univariate associations. Results: The median follow-up for patients who were censored (n = 15, 26%) was 15 mos. (interquartile range (IQR): 9 -21). Most patients were diagnosed with lung (n = 27, 47%) or breast cancer (n = 22, 38%). The median CNS PFS and OS were 6 mos. (IQR: 3 – 9) and 8 mos. (IQR: 5 – 18), respectively. Of the 49 patients with pre-pCSI CSF-CTCs analyzed, CSF-CTCs were identified in 43 (88%). Pre-pCSI CSF-CTC&lt; 53/3mL was associated with improved CNS PFS (11.8 vs 6.0 mos., p = 0.01), and a trend toward improved OS (16.7 vs 7.7 mos., p = 0.08). On pre-pCSI MRI, patients with parenchymal brain metastases (n = 33, 57%) had worse OS (6.7 vs 12.7 mos., p = 0.01) but not CNS PFS. Patients with both brain and spine LM (n = 42, 72%) compared to those only one site or no visible disease (n = 16, 28%) showed worse CNS PFS (5.8 vs 7.5 mos., p = 0.03) and OS (7.7 vs 16.7 mos., p = 0.05). In a multivariate model, pre-pCSI CSF-CTC was significantly associated with CNS PFS (p = 0.03) while brain and spine LM on MRI was not (p = 0.20) No patient had an increase in CSF-CTC immediately post-pCSI, and in those with both detectable pre-pCSI CSF-CTCsand a post-pCSImeasurement(n = 29, 50%), the median decrease at nadir was 37/3mL (range: 0-200) occurring at a median of 1.6 mos. (range: 0.5 -5.2). A decrease in CSF-CTC &gt; 37/3mL was associated with improved CNS PFS (7.1 vs 4.4 mos., p = 0.04) but not OS (12.5 vs.7.7 mos., p = 0.2). Conclusions: Proton CSI is an effective treatment for patients with solid tumor LM and can result in prolonged disease control in some patients. Lower CSF-CTC count prior to pCSI and larger changes after pCSI are predictive of survival outcomes, arguing for early pCSI intervention for solid tumor LMD. Early treatment escalation after pCSI can be considered for patients with high pre-pCSI CSF-CTC and a smaller nadir post-pCSI.

Quantitative cerebrospinal fluid circulating tumor cells are a potential biomarker of response for proton craniospinal irradiation for leptomeningeal metastasis.

BackgroundLeptomeningeal metastasis (LM) involves cerebrospinal fluid (CSF) seeding of tumor cells. Proton craniospinal irradiation (pCSI) is potentially effective for solid tumor LM. We evaluated whether circulating tumor cells (CTCs) in the CSF (CTCCSF), blood (CTCblood), and neuroimaging correlate with outcomes after pCSI for LM.MethodsWe describe a single-institution consecutive case series of 58 patients treated with pCSI for LM. Pre-pCSI CTCs, the change in CTC post-pCSI (Δ CTC), and MRIs were examined. Central nervous system progression-free survival (CNS-PFS) and overall survival (OS) from pCSI were determined using Kaplan Meier analysis, Cox proportional-hazards regression, time-dependent ROC analysis, and joint modeling of time-varying effects and survival outcomes.ResultsThe median CNS-PFS and OS were 6 months (IQR: 4–9) and 8 months (IQR: 5–13), respectively. Pre-pCSI CTCCSF < 53/3mL was associated with improved CNS-PFS (12.0 vs 6.0 months, P < .01). Parenchymal brain metastases (n = 34, 59%) on pre-pCSI MRI showed worse OS (7.0 vs 13 months, P = .01). Through joint modeling, CTCCSF was significantly prognostic of CNS-PFS (P < .01) and OS (P < .01). A Δ CTC-CSF≥37 cells/3mL, the median Δ CTC-CSF at nadir, showed improved CNS-PFS (8.0 vs 5.0 months, P = .02) and further stratified patients into favorable and unfavorable subgroups (CNS-PFS 8.0 vs 4.0 months, P < .01). No associations with CTCblood were found.ConclusionWe found the best survival observed in patients with low pre-pCSI CTCCSF and intermediate outcomes for patients with high pre-pCSI CTCCSF but large Δ CTC-CSF. These results favor additional studies incorporating pCSI and CTCCSF measurement earlier in the LM treatment paradigm.

Cytoplasm protein GFAP magnetic beads construction and application as cell separation target for brain tumors

BackgroundIt is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice. In contrast to the past use of epithelial EpCAM as CTCs separation target, a cytoplasm protein of GFAP antibody was first selected to construct highly-sensitive immunomagnetic liposome beads (IMLs). The validation and efficiency of this system in capturing CTCs for brain tumors were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed.ResultsOur data show that CTCs can be successfully isolated from CSF and blood samples from 32 children with brain tumors. The numbers of CTCs in CSF were significantly higher than those in blood. The level of CTCs in CSF was related to the type and location of the tumor rather than its stage. The higher the CTCs number is, the more possibly the patient will suffer from poor prognosis. Genetic testing in GFAP CTC-DNA by sanger sequencing, q-PCR and NGS methods indicated that the isolated CTCs (GFAP+/EGFR+) are the related tumor cell. For example, the high expression of NPR3 gene in CSF CTCs was consistent with that of tumor tissue.ConclusionsThe results indicated that GFAP-IML CTCs isolation system, combined with an EGFR immunofluorescence assay of antitumor marker, can serve as a brand-new method for the identification of CTCs for brain tumors. Via lumbar puncture, a minimally invasive procedure, this technique may play a significant role in the clinical diagnosis and drug evaluation of brain tumors.

RADT-05. CLINICAL OUTCOMES OF PROTON CRANIOSPINAL IRRADIATION FOR PATIENTS WITH LEPTOMENINGEAL CARCINOMATOSIS

Abstract Leptomeningeal carcinomatosis (LC) is a devastating complication of metastatic tumors. Radiotherapy (RT) is integral to LC treatment, and proton craniospinal irradiation (CSI) may make RT more effective by targeting the entire central nervous system (CNS) compartment. We evaluated outcomes in patients treated with proton CSI for LC. We identified 56 patients treated with proton CSI for LC between 2018 and 2020 at our institution. Data on patient demographics, disease and treatment history, cerebrospinal fluid circulating tumor cells (CSF CTCs), and gene alterations were collected. Kaplan Meier analysis and Cox regression models were used to compare correlates with CNS time to progression (TTP), CNS progression-free survival (PFS) and overall survival (OS). Most patients had non-small cell lung cancer (NSCLC, n=27, 48%) or breast cancer (n=21, 38%). The median age was 58 (30-77), and median KPS was 80 (60-90). The median RT dose was 30Gy (25-36). The median follow-up was 12 months (1-22), with 26 (46%) patients alive at the last follow-up. Of 35 (63%) patients who progressed, 6 (11%) progressed in the CNS, 13 (23%) progressed systemically, and 16 (29%) progressed in both. The median TTP, PFS and OS was 8 months (1-21), 6 months (1-21) and 8 months (1-22), respectively. No difference in PFS (7 vs. 6 months, p=0.6) and OS (8 vs. 7 months, p=0.3) was observed between patients with NSCLC and breast cancer. Of patients alive at 3 months, 79% showed stable or improved functional status after CSI. Decreased CSF CTCs immediately post CSI had significantly improved PFS (8 vs. 5 months with no CTC decrease, HR=0.3, p=0.02). Lastly, we identified genetic correlates with survival outcomes. Proton CSI appears to be a promising treatment for LC in select patients, resulting in prolonged CNS disease control and survival. A randomized trial is currently underway to assess its efficacy prospectively.

Cerebrospinal fluid circulating tumor cells as a quantifiable measurement of leptomeningeal metastases in patients with HER2 positive cancer

PurposeThe CellSearch® system has been used to identify circulating tumor cells (CTCs) in cerebrospinal fluid (CSF) to diagnose leptomeningeal metastasis (LM) in patients with epithelial cancers. Using this system, we prospectively explored sequential CSF CTC enumeration in patients with LM from HER2+ cancers receiving intrathecal (IT) trastuzumab to capture dynamic changes in CSF CTC enumeration.MethodsCSF from patients enrolled in an IRB-approved phase I/II dose escalation trial of IT trastuzumab for LM in HER2+ cancer (NCT01325207) was obtained on day 1 of each cycle and was evaluated by the CellSearch® platform for CTC enumeration. The results were correlated with CSF cytology from the same sample, along with clinical and radiographic response.ResultsFifteen out of 34 patients with HER2+ LM were enrolled in CSF CTC analysis; 14 were women. Radiographic LM was documented in 14 (93%) patients; CSF cytology was positive in 6 (40%) and CSF CTCs were identified in 13 (87%). Median CSF CTC was 22 CTCs (range 0–200 +) per 3 ml. HER2/neu expression analysis of CTCs was performed in 8 patients; 75% had confirmed expression of HER2/neu positivity in CSF and HER2/neu expression was absent in 25%. Four of 10 patients received 7 or more cycles of IT trastuzumab; in 3 of these patients, increase in CSF CTCs enumeration from baseline was detected 2–3 months prior to changes seen on MRI, and while CSF cytology remained negative.ConclusionOur study demonstrates that enumeration of CSF CTCs may provide dynamic, quantitative assessment of tumor burden in the central nervous system compartment during treatment for LM and prior to changes on MRI or CSF cytology.Trial Registration: Clinicaltrials.gov: NCT01325207; registered March 29th, 2011.

CMET-21. A PHASE IB STUDY OF PROTON CRANIOSPINAL IRRADIATION FOR THE TREATMENT OF SOLID TUMOR LEPTOMENINGEAL METASTASES

Abstract Leptomeningeal metastases (LM) is associated with limited survival and treatment options for patients with solid tumor malignancies. While focal radiotherapy is effective for local palliation, it lacks durability due to cerebrospinal fluid (CSF) tumor cells reseeding. Craniospinal irradiation (CSI) in contrast treats the entire central nervous system (CNS) compartment thus potentially improves disease control. We performed a phase IB study of proton CSI using 30CGE in 10 fractions for treating solid tumor LM (NCT03520504). The primary end point is dose-liming toxicity (DLT) within 1 month of treatment. Clinical outcomes and CSF circulating tumor cells (CTCs) by CellSearch® were evaluated. Overall (OS) and progression-free survival (PFS) were assessed using Kaplan-Meier estimates. Of the 24 patients enrolled, 4 were excluded from analysis: 3 did not complete treatment due to CNS or systemic disease progression (PD), and 1 completed treatment outside study window. The majority of patients had metastatic lung (55%) and breast (30%) malignancies. At this submission, the median follow-up was 2.8 months (0.5–9.4 months). No DLT was observed. Treatment-related grade 2+ toxicities were: fatigue (G2=43%), anorexia (G2=5%), anemia (G2=10%), thrombocytopenia (G3=5%, G2=10%), and leukopenia (G3=14%, G2=14%). Median survival is not reached with 17 patients (85%) alive with stable/improved LM including 2 with durable CNS control for &gt;9 months, 1 patient alive with CNS and systemic PD at 3.7 months, 2 patients died at 3.6 and 5.1 months with CNS and systemic PD. At 3-month, OS and PFS were 100% and 90% (95% CI 100–72%), respectively. In the 13 patients with CSF CTCs evaluation, decreased quantifiable CTCs at 1-month after proton CSI correlated significantly with improved CNS PFS (log-rank p=0.014). These early findings suggest that proton CSI is a safe and potentially effective treatment for patients with solid tumor LM. CSF biomarkers assessment is needed to better elucidate predictors of response.

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

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. 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. 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. 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.

P05.10 Prospective evaluation of cerebrospinal fluid circulating tumor cells (CSF CTC) in patients with HER2 positive cancers and leptomeningeal metastases receiving treatment with intrathecal trastuzumab

P05.10 Prospective evaluation of cerebrospinal fluid circulating tumor cells (CSF CTC) in patients with HER2 positive cancers and leptomeningeal metastases receiving treatment with intrathecal trastuzumab

Detection of Driver and Resistance Mutations in Leptomeningeal Metastases of NSCLC by Next-Generation Sequencing of Cerebrospinal Fluid Circulating Tumor Cells.

Purpose: Leptomeningeal metastases are more common in non-small cell lung cancer (NSCLC) with EGFR mutations. The diagnosis is difficult by traditional imaging only, and leads to poor understanding of resistance mechanisms of leptomeningeal metastases.Experimental Design: We compared the CellSearch Assay, the Thinprep cytologic test (TCT), and brain magnetic resonance imaging (MRI) in 21 NSCLC patients with suspected leptomeningeal metastases. Next-generation sequencing that included 416 cancer-associated genes was also performed on cerebrospinal fluid circulating tumor cells (CSFCTC) of 19 patients.Results: Twenty-one patients were diagnosed with leptomeningeal metastases, and CSFCTCs were captured by CellSearch in 20 patients (median, 969 CSFCTCs/7.5 mL; range, 27-14,888). CellSearch had a sensitivity of 95.2% for leptomeningeal metastases diagnosis, which was higher than that of TCT (12/21, 57.1%), MRI (10/21, 47.6%), and MRI plus TCT (19/21, 90.5%), respectively. CTCs were found only in 5 of 14 patients (median, 2 CTCs/7.5 mL; range, 2-4), which was a much lower ratio than CSFCTCs. Genetic profiles of CSFCTCs were highly concordant with molecular mutations identified in the primary tumor (17/19, 89.5%). The resistance gene EGFR T790M was detected in 7 of 9 patients with extracranial lesions, but was detected in only 1 of 14 CSFCTC samples. Other potential resistant mutations, such as MET amplification and ERBB2 mutation, were also identified in CSFCTCs.Conclusions: CSFCTCs captured by CellSearch may be a more sensitive and effective way to diagnose leptomeningeal metastases, and may serve as a liquid biopsy medium for gene profiles in NSCLC patients with leptomeningeal metastases. Clin Cancer Res; 23(18); 5480-8. ©2017 AACR.

1307PD - Detection of driver and resistance mutations in leptomeningeal metastases of NSCLC by next-generation sequencing of cerebrospinal fluid circulating tumor cells

Background: Leptomeningeal metastases (LM) are more common in non-small cell lung cancer (NSCLC) with EGFR mutations. The diagnosis is difficult by traditional imaging only, and leads to poor understanding of resistance mechanisms of LM. Methods: We compared the CellSearch Assay™, the Thinprep cytologic test (TCT), and brain magnetic resonance imaging (MRI) in 21 NSCLC patients with suspected LM. Next-Generation sequencing that included 416 cancer-associated genes was also performed on cerebrospinal fluid circulating tumor cells (CSFCTCs) of 19 patients. Results: Twenty-one patients were diagnosed with LM, and CSFCTCs were captured by CellSearch in 20 patients (median, 969 CSFCTCs/7.5 mL; range, 27–14,888). CellSearch had a sensitivity of 95.2% for LM diagnosis, which was higher than that of TCT (12/21, 57.1%), MRI (10/21, 47.6%), and MRI plus TCT (19/21, 90.5%), respectively. CTCs were found only in 5 of 14 patients (median, 2 CTCs/7.5 mL; range, 2–4), which was a much lower ratio than CSFCTCs. Genetic profiles of CSFCTCs were highly concordant with molecular mutations identified in the primary tumor (17/19, 89.5%). The resistance gene EGFR T790M was detected in 7 of 9 patients with extracranial lesions, but was only detected in 1 of 14 CSFCTCs samples. Other potential resistant mutations such as MET amplification and ERBB2 mutation were also identified in CSFCTCs.Table1307PDPatient No.Primary gene profileTargeted therapy before LMRebiopsy gene profileCSFCTCs gene profile (NGS)1EGFR L858R (lung)ErlotinibEGFR L858R (lung, ARMS); MET(IHC):80% (3+)EGFR L858R2bEGFR del19 (lymph node)IcotinibUAEGFR del19; EGFR T790M3EML4-ALK (lung)CrizotinibEML4-ALK (pleural effusion)EML4-ALK; MET amplification4EGFR 20INS (lung)NoneUAEGFR 20INS5EGFR del19 (pleura)GefitinibEGFR del19 and T790M (lung)EGFR del19; EGFR amplification6EGFR L858R (lung)ErlotinibEGFR L858R and T790M (lung)EGFR L858R7EGFR, ALK (WT)NoneSnapshot, MET, KIT (WT)Common driver gene (WT)9EGFR del19 (pleura)GefitinibEGFR del19 and T790M (liver);MET: 100% (3+) (liver)EGFR del19; ERBB2 exon8 T328fs; ROS1 exon7 W215X10EGFR L858R (lung)Gefitini, erlotinibEGFR del19 and T790M (lung)EGFR del1911EGFR L858R (lung)Gefitini, erlotinibUAEGFR L858R12EGFR L858R (lung)GefitinibEGFR L858R and T790M (plasma)Common driver gene (WT)13EML4-ALK (lung)CrizotinibUAEML4-ALK14EGFR del19 (lung)Erlotinib, afatinibUAEGFR del19; MET amplification15EGFR del19 (lung)IcotinibUAEGFR del1916EGFR L858R (lung)NoneEGFR L858R (lymph node)EGFR and ALK (WT); RET exon4 V253E17EGFR L861Q (lymph node)ErlotinibUAEGFR L861Q; EGFR del1918cEGFR L858R (lung)GefitinibEGFR L858R and T790M (lung)EGFR L858R; PIK3CA exon2 N107S; MET exon11 F839L19EGFR L858R (lung)GefitinibEGFR L858R and T790M (lung)EGFR L858R21EGFR L858R (lung)GefitinibUAEGFR L858R Open table in a new tab Conclusions: CellSearch could be a more sensitive method for detecting tumor cells in CSF, and potentially provides earlier diagnosis of LM. More importantly, CSFCTCs could be an important and new way of “liquid biopsy” for genetic profiles of metastatic tumor cells in LM patients of NSCLC. Legal entity responsible for the study: Yi-Long Wu Funding: Geneseeq Biotechnology, Inc., Nanjing, China Disclosure: All authors have declared no conflicts of interest.

Cerebrospinal fluid circulating tumor cells: a novel tool to diagnose leptomeningeal metastases from epithelial tumors.

Diagnosis of leptomeningeal metastasis (LM) remains challenging due to low sensitivity of CSF cytology and infrequent unequivocal MRI findings. In a previous pilot study, we showed that rare cell capture technology (RCCT) could be used to detect circulating tumor cells (CTC) in the CSF of patients with LM from epithelial tumors. To establish the diagnostic accuracy of CSF-CTC in the diagnosis of LM, we applied this technique in a distinct, larger cohort of patients. In this institutional review board-approved prospective study, patients with epithelial tumors and clinical suspicion of LM underwent CSF-CTC evaluation and standard MRI and CSF cytology examination. CSF-CTC enumeration was performed through an FDA-approved epithelial cell adhesion molecule-based RCCT immunomagnetic platform. LM was defined by either positive CSF cytology or imaging positive for LM. ROC analysis was utilized to define an optimal cutoff for CSF-CTC enumeration. Ninety-five patients were enrolled (36 breast, 31 lung, 28 others). LM was diagnosed in 30 patients (32%) based on CSF cytology (n = 12), MRI findings (n = 2), or both (n = 16). CSF-CTC were detected in 43/95 samples (median 19.3 CSF-CTC/mL, range 0.3 to 66.7). Based on ROC analysis, 1 CSF-CTC/mL provided the best threshold to diagnose LM, achieving a sensitivity of 93%, specificity of 95%, positive predictive value 90%, and negative predictive value 97%. We defined ≥1 CSF-CTC/mL as the optimal cutoff for diagnosis of LM. CSF-CTC enumeration through RCCT is a robust tool to diagnose LM and should be considered in the routine LM workup in solid tumor patients.