Tumor Cells In Cerebrospinal Fluid Research Articles

A Microfluidic, Multi-Antibody Cell Capture Method to Evaluate Tumor Cells in Cerebrospinal Fluid in Patients With Suspected Leptomeningeal Metastases.

Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors. It has a grave prognosis without urgent treatment. Standard of care methodologies to diagnose LMD include CSF cytology, magnetic resonance imaging, and clinical evaluation. These methods offer limited sensitivity and specificity for the evaluation of LMD. Here, we describe the analytic performance characteristics of a microfluidic-based tumor cell enrichment and detection assay optimized to detect epithelial cells in CSF using both contrived samples as well as CSF from patients having suspected or confirmed LMD from carcinomas. To demonstrate the feasibility of using a microfluidic, multi-antibody cell capture assay to identify and quantify tumor cells in CSF. An artificial CSF solution was spiked with 34 different human carcinoma cell lines at different concentrations and assayed for the ability to detect tumor cells to assess analytic accuracy. Two cell lines were selected to assess linearity, intra-assay precision, interinstrument precision, and sample stability. Clinical verification was performed on 65 CSF specimens from patients. Parameters assessed included the number of tumor cells, coefficient of variation percentage, and percentage of tumor cell capture (TCC). Among contrived samples, average tumor cell capture ranged from 50% to 82% (261 of 522; 436 of 531), and coefficients of variation ranged from 7% to 67%. The cell capture assay demonstrated a sensitivity of 92% and a specificity of 95% among clinical samples. This assay demonstrated the ability to detect and enumerate epithelial cells in contrived and clinical specimens in an accurate and reproducible fashion. The use of cell capture assays in CSF may be useful as a sensitive test for the diagnosis and longitudinal monitoring of LMD from solid tumors.

The HER2 flip-HER2 amplification of tumor cells in the cerebrospinal fluid of breast cancer patients with leptomeningeal disease: implications for treating the LM tumor with anti-HER2 therapy.

CNSide is a platform that detects and characterizes tumor cells in the cerebrospinal fluid (CSF) of patients with leptomeningeal disease (LMD). The platform was validated per College of American Pathologists (CAP) and Clinical Laboratories Improvement Amendment (CLIA) guidelines and run as a commercial Laboratory Developed Test (LDT) at Biocept in San Diego, CA. The platform allows CSF tumor cell (CSF-TC) enumeration and biomarker characterization by fluorescent in situ hybridization (FISH). We performed a multicenter retrospective chart review of HER2 FISH CNSide test results that were commercially ordered on 26 patients by physicians for LMD breast cancer patients between April 2020 and October 2022. We show that HER2 is amplified on CSF tumor cells in 62% (16/26) of LMD breast cancer patients. 10/26 (38%) patients had discordant HER2-positivity between the primary tumor tissue and CSF-TC; of these, 35% (9/26) of the patients displayed HER2 amplification on the CSF-TCs, however were categorized as HER2 negative on the primary tumor. Of the 27% (7/26) patients with a HER2 positive primary tumor, one patient showed a HER2 negative LMD tumor. Two patients, 8% (2/26) had a HER2 equivocal primary tumor; of these, one demonstrated a HER2 negative, and one a HER2 positive LMD tumor. Serial analysis (at least 4 longitudinal tests) of HER2 status of the CSF-TC throughout therapy was available for 14 patients and demonstrated that HER2 status of the LMD changed in 29% (4/14) during their treatment course and impacted care decisions. Our data suggests that CSF-TC HER2 FISH analysis in LMD breast cancer patients may be discordant to the primary tumor sample and the discovery of HER2 positivity in the CSF may open doors to anti-HER2 targeted therapy options for LMD patients.

Abstract PO4-20-11: Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay

Abstract A 72-year-old female presented to the emergency department with sudden onset of imbalance and cognitive decline. Two years ago, she was diagnosed with stage III estrogen receptor (ER) positive, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative (immunohistochemistry score: 0) invasive breast cancer. She subsequently underwent lumpectomy, adjuvant radiation and chemotherapy. She had been taking adjuvant letrozole at the time of presentation. Magnetic resonance imaging (MRI) of the brain showed obstructive hydrocephalus without any parenchymal lesion or leptomeningeal enhancement. MRI of spine, computerized tomography of chest/abdomen/pelvis and bone scan were unrevealing except a left adrenal mass which was equivocal for metastasis. Cerebrospinal fluid (CSF) analysis showed rare malignant cells. Based on the morphology of malignant cells and the patient’s prior history of breast cancer, the diagnosis of leptomeningeal disease with breast primary was made. There were insufficient cells for additional studies. CNSide, a microfluidic platform that uses antibody capture method to detect CSF tumor cells, showed 4678 tumor cells per milliliter (ml) of CSF. Immunocytochemistry of these cells showed that the cells were ER negative (criterion: < 2.6% ER positive cells) and programmed cell death ligand 1 (PD-L1) negative (criterion: < 3.4% PD-L1 positive cells). Fluorescence in situ hybridization (FISH) testing showed 22% of tumor cells with HER2 amplification (either HER2 to CEP17 ratio >/= 2.0 or HER2 copy number >/= 6). She underwent ventriculoperitoneal (VP) shunt placement with complete recovery of her neurological symptoms. Five weeks later, she was started on trastuzumab deruxtecan (T-DXd) mainly because of the HER2 FISH results from CNSide. She did not receive central nervous system(CNS) irradiation due to complete resolution of neurological symptoms with VP shunt placement. After two cycles of T-DXd, MRI brain showed mild leptomeningeal enhancement along bilateral superior cerebellar sulci. Positron emission tomography/computed tomography(PET/CT) scan showed a new sclerotic rib focus, thought to represent treatment response. It also showed decrease in size of the adrenal mass. The adrenal mass was hypermetabolic on PET, consistent with malignancy. CSF cytology was negative, while CSF tumor cell count significantly reduced to 6.72 cells/ml using CNSide. There were no new neurological symptoms. Despite MRI brain findings, she was continued on T-DXd given overall favorable clinical, radiological and CSF data. After 7th cycle of T-DXd, repeat CSF cytology continued to remain negative, CNSide CSF tumor cell count decreased further to 6.27 cells/ml, MRI brain showed interval resolution of prior leptomeningeal enhancement, CT scan showed further decrease in size of the adrenal mass (baseline 5.4cm to 3cm), stable rib sclerotic lesion and no additional metastatic lesion. To date, she has undergone eight cycles of T-DXd which has effectively controlled her disease for a period of seven months. She has not needed VP shunt revision and continues to do well. To our knowledge, this is the first case demonstrating the activity of T-DXd against CSF tumor cells where a small subset exhibited HER2 amplification. This case also highlights the benefits of using advanced and highly sensitive CSF assays to diagnose and surveil leptomeningeal disease and to assess HER2 signaling. This case is well-timed especially in the era of next-generation, CNS penetrating, potent HER2-directed antibody-drug conjugate like T-DXd which can mount antitumor activity even with minimal HER2 expression in tumors. Citation Format: Sarah Stanford, Zeni Kharel, Lauryn Hemminger, Tyler Schmidt, Sara Hardy, Jason Zittel, Nimish Mohile, Ajay Dhakal. Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-11.

Prognostic value of cerebrospinal fluid tumor cell count in leptomeningeal disease from solid tumors.

Treatment decisions for leptomeningeal disease (LMD) rely on patient risk stratification, since clinicians lack objective prognostic tools. The introduction of rare cell capture technology for identification of cerebrospinal fluid tumor cells (CSF-TCs), such as CNSide assay, improved the sensitivity of LMD diagnosis, but prognostic value is unknown. This study assesses the prognostic value of CSF-TC density in patients with LMD from solid tumors. We conducted a retrospective cohort study of patients with newly diagnosed or previously treated LMD from a single institution who had CNSide assay testing for CSF-TCs from 2020 to 2023. Univariable and multivariable survival analyses were conducted with Cox proportional-hazards modeling. Maximally-selected rank statistics were used to determine an optimal cutpoint for CSF-TC density and survival. Of 31 patients, 29 had CSF-TCs detected on CNSide. Median (interquartile range [IQR]) CSF-TC density was 67.8 (4.7-639) TCs/mL. CSF cytology was positive in 16 of 29 patients with positive CNSide (CNSide diagnostic sensitivity = 93.5%, negative predictive value = 85.7%). Median (IQR) survival from time of CSF-TC detection was 176 (89-481) days. On univariable and multivariable analysis, CSF-TC density was significantly associated with survival. An optimal cutpoint for dichotomizing survival by CSF-TC density was 19.34 TCs/mL. The time-dependent sensitivity and specificity for survival using this stratification were 76% and 67% at 6 months and 65% and 67% at 1 year, respectively. CSF-TC density may carry prognostic value in patients with LMD from solid tumors. Integrating CSF-TC density into LMD patient risk-stratification may help guide treatment decisions.

Keeping a track on leptomeningeal disease in non-small cell lung cancer: A single-institution experience with CNSideTM.

Leptomeningeal disease (LMD) is a devastating complication for patients with advanced cancer. Diagnosis and monitoring the response to therapy remains challenging due to limited sensitivity and specificity of standard-of-care (SOC) diagnostic modalities, including cerebrospinal fluid (CSF) cytology, MRI, and clinical evaluation. These hindrances contribute to the poor survival of LMD patients. CNSide is a CLIA-validated test that detects and characterizes CSF-derived tumor cells and cell-free (cf) DNA. We performed a retrospective analysis on the utility of CNSide to analyze CSF obtained from advanced non-small cell lung cancer (aNSCLC) patients with suspected LMD treated at the Huntsman Cancer Institute in Salt Lake City, UT. CNSide was used to evaluate CSF from 15 patients with aNSCLC. CSF tumor cell quantification was performed throughout treatment for 5 patients. CSF tumor cells and cfDNA were characterized for actionable mutations. In LMD-positive patients, CNSide detected CSF tumor cells in 88% (22/25) samples versus 40% (10/25) for cytology (matched samples). CSF tumor cell numbers tracked response to therapy in 5 patients where CNSide was used to quantify tumor cells throughout treatment. In 75% (9/12) of the patients, genetic alterations were detected in CSF, with the majority representing gene mutations and amplifications with therapeutic potential. The median survival for LMD patients was 16.1 m (5.2-NR). We show that CNSide can supplement the management of LMD in conjunction with SOC methods for the diagnosis, monitoring response to therapy, and identifying actionable mutations unique to the CSF in patients with LMD.

Comparison of mutation detection rate between cell pellet DNA and cell-free DNA from cerebrospinal fluid in patients with advanced lung cancer.

e21014 Background: Liquid biopsy of cerebrospinal fluid (CSF) has been approved to be a promising strategy for molecular pathology testing in patients with lung cancer. Numerous studies showed that CSF-derived DNA can recapitulate the molecular characteristics of metastatic lung cancer. However, differences in mutation detection rate and the overall mutational profile between cell pellet DNA and supernatant cell-free DNA (cfDNA) from CSF in patients with lung cancer have rarely been reported. Methods: Matched CSF cell pellet and supernatant sample sets were selected from 51 lung cancer patients in our sequencing center. CSF was centrifuged (1000g, 10 min) to separate the cell pellet and supernatant components. Cellular DNA and cfDNA were extracted and underwent library preparation procedures for next-generation sequencing respectively. Cut adapter, mapping, fusion calling and cnv calling were performed for sequencing data analysis. Results: At the sample level, gene mutation could be detected in both CSF cell pellet and cfDNA, and the positive detection rate of gene mutation was consistent. The positive percent agreement (PPA) of CSF cfDNA sequencing compared with CSF cell pellet DNA was 93%. At the genetic variation level, 223 gene mutation sites were detected, and the positive rate of gene mutation in the CSF cfDNA (32.51%) was significantly higher than that in the CSF cell pellet (10.74%, p＜0.01). Additionally，the variation rate of less than 1% was defined as the low-frequency mutation range. Results showed that variant allele frequency (VAF) in CSF cfDNA were remarkably higher than those in the CSF cell pellet in both low-and high-frequency mutation ranges ( p＜0.01, p＜0.01). Conclusions: Both cell pellet DNA and cfDNA isolated from CSF can be used for molecular subtyping of lung cancer. Given that CSF is inevitably contaminated with blood cells in clinical procedures, the dynamic range of tumor cells in CSF could be quite large. CSF cfDNA better recapitulate the mutational status and is recommended to consider first for liquid biopsy in advanced lung cancer with brain metastases or suspicious brain metastases. Key words: Next-generation sequencing; CSF; cell pellet DNA; cfDNA; advanced lung cancer.

Abstract P1-05-23: OPTIMIZING THE DIAGNOSIS OF LEPTOMENINGEAL METASTASES IN BREAST CANCER PATIENTS BY CIRCULATING TUMOR CELLS AND CIRCULATING TUMOR DNA

Abstract Introduction: Leptomeningeal metastases (LM) occur in approximately 5% of patients with breast cancer, negatively impacting their prognosis. Reliable diagnosis of LM is challenging, but timely diagnosis could lead to more treatment options and consequently a better prognosis. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in cerebrospinal fluid (CSF) are potential methods to improve the diagnosis of LM. In this prospective study we compared cytology (current gold standard for CSF analysis in diagnosing LM) with CTC enumeration and ctDNA detection in CSF for diagnosing LM in patients with breast cancer. Methods: In a prospective study in patients with clinical suspicion of LM who were planned to undergo a diagnostic lumbar puncture (LP) for CSF cytology the presence of CTCs/ctDNA in CSF was compared to the presence of tumor cells in CSF by cytology. CTC enumeration was performed using the FDA approved CellSearch System (Menarini). The detection of ctDNA was performed using the mFAST-Seqs method, which detects aneuploidy throughout the genome by selective amplification of long interspaced nuclear element sequences (LINE-1 elements). In a control group consisting of 15 patients with a hematologic malignancy with suspicion of LM, 2 cases had 1 CTC in their CSF, therefore the cutoff for a positive CTC test was set at 2 CTCs. Results: In total 80 patients with breast cancer were included from January 2016 till January 2022. In 16 out of 80 patients CSF cytology was positive of whom 14 (87.5%) had a positive CTC test. From the 2 patients in which the CTC test was negative, one patient had 1 CTC detected in CSF and for both patients a lower volume of CSF was available for the CTC test compared to the volume used for cytology. In 2 of the 16 cytology positive patients, who underwent a second LP, CTCs were positive in the CSF from the first LP, while cytology was only positive in the CSF of the second LP. Of the 64 patients with a negative cytology, 7 (10.9%) patients had a positive CTC test. Two of these 7 patients had typical symptoms of LM, a suspicious MRI and a course of disease matching with LM according to the neuro-oncologist. Overall survival (OS) was shorter in patients with a positive CTC test compared to patients with a negative test (median OS: 19.2 months vs. 3.4 months, log-rank test, p=0.007). When only patients with a negative cytology were included in this analysis, OS remains significantly shorter in the group with a positive CTC test (n=7) compared to the group with the negative CTCs (n=57) (median OS 19.3 months vs. 3.5 months, long-rank test, p=0.003). Additionally, ctDNA detection in CSF was compared to CSF cytology in 51 patients, of which 11 had a positive CSF cytology result. For 8 of these 11 patients ctDNA was detected (72.2%) while 1 of the 40 patients with a negative cytology had ctDNA. Discussion: Both CTC enumeration and ctDNA detection can be used to detect LM in CSF. CTC detection could even improve timely diagnosis of LM in patients with breast cancer. However, the added value of ctDNA seems less evident. Citation Format: Elisabeth M Jongbloed, Lindsay Angus, Martin J van den Bent, Joost LM Jongen, John WM Martens, Ngoc M Van, Vanja de Weerd, Carolien HM van Deurzen, Jaco Kraan, Saskia M Wilting, Agnes Jager. OPTIMIZING THE DIAGNOSIS OF LEPTOMENINGEAL METASTASES IN BREAST CANCER PATIENTS BY CIRCULATING TUMOR CELLS AND CIRCULATING TUMOR DNA [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-23.

Early Detection of Leptomeningeal Metastases Among Patients Undergoing Spinal Stereotactic Radiosurgery

PurposeThe management of patients with advanced solid malignancies increasingly uses stereotactic body radiation therapy (SBRT). Advanced cancer patients are at risk for developing leptomeningeal metastasis (LM), a fatal complication of metastatic cancer. Cerebrospinal fluid (CSF) is routinely collected during computed tomography (CT) myelography for spinal SBRT planning, offering an opportunity for early LM detection by CSF cytology in the absence of radiographic LM or LM symptoms (subclinical LM). This study tested the hypothesis that early detection of tumor cells in CSF in patients undergoing spine SBRT portends a similarly poor prognosis compared with clinically overt LM. Methods and MaterialsWe retrospectively analyzed clinical records for 495 patients with metastatic solid tumors who underwent CT myelography for spinal SBRT planning at a single institution from 2014 to 2019. ResultsAmong patients planned for SBRT, 51 (10.3%) developed LM. Eight patients (1.6%) had subclinical LM. Median survival with LM was similar between patients with subclinical versus clinically evident LM (3.6 vs 3.0 months, P = .30). Patients harboring both parenchymal brain metastases and LM (29/51) demonstrated shorter survival than those with LM alone (2.4 vs 7.1 months, P = .02). ConclusionsLM remains a fatal complication of metastatic cancer. Subclinical LM detected by CSF cytology in spine SBRT patients has a similarly poor prognosis compared with standardly detected LM and warrants consideration of central nervous system-directed therapies. As aggressive local therapies are increasingly used for metastatic patients, more sensitive CSF evaluation may further identify patients with subclinical LM and should be evaluated prospectively.

1063P Comprehensive genomic profiling of leptomeningeal metastases on NSCLC patients through circulating tumor DNA in cerebrospinal fluid

Leptomeningeal metastases occurred in more than 3% of patients diagnosed with NSCLC, resulting in poor clinical outcomes. The cerebrospinal fluid (CSF) was a direct liquid biopsy for pathological diagnosis of leptomeningeal metastases. However, traditional clinical methods of detecting tumor cells in CSF showed limited sensitivity. Meanwhile, the unique genomic aberrations of leptomeningeal metastases remained unclear.

Detection of Circulating Tumor Cells in Cerebrospinal Fluid of Patients with Suspected Breast Cancer Leptomeningeal Metastases: A Prospective Study.

The diagnosis of breast cancer (BC)-related leptomeningeal metastases (LM) relies on the detection of tumor cells in cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, the sensitivity of this technique is low. Our goal was to evaluate whether circulating tumor cell (CTC) detection in CSF using the CellSearch® system could be used for LM diagnosis. This prospective, monocentric study included adult patients with suspected BC-related LM. The clinical sensitivity and specificity of CTC detection in CSF for LM diagnosis were calculated relative to conventional CSF cytology. Forty-nine eligible patients were included and 40 were evaluable (CTC detection technical failure: n = 8, eligibility criteria failure: n = 1). Cytology was positive in 18/40 patients. CTCs were detected in these 18 patients (median: 5824 CTC, range: 93 to 45052) and in 5/22 patients with negative cytology (median: 2 CTC, range: 1 to 44). The detection of ≥1 CSF CTC was associated with a clinical sensitivity of 100% (95% CI, 82.4-100) and a specificity of 77.3% (95% CI, 64.3-90.3) for LM diagnosis. HER2+ CTCs were detected in the CSF of 40.6% of patients with HER2- BC (median: 500 CTC, range: 13 to 28 320). The clinical sensitivity of CTC detection in CSF with the CellSearch® system for LM diagnosis is higher than that of CSF cytology. CTC detection in patients with negative cytology, however, must be further investigated. The finding of HER2+ CTCs in patients with HER2- BC suggests that the HER2 status of LM should be evaluated to increase the treatment opportunities for these patients.

Investigation of white blood cell characteristics in cerebrospinal fluid samples at pediatric brain tumor diagnosis.

The role of white blood cells (WBC) in the pediatric central nervous system (CNS) tumor microenvironment is incompletely defined. We hypothesized that the WBC profile in cerebrospinal fluid (CSF) correlates with the presence of tumor cells and prognosis in pediatric CNS tumors, as well as other patient and disease characteristics, and differs by tumor type, thus giving insight into the tumor immune response. We conducted a retrospective analysis of CSF WBC profiles at CNS tumor diagnosis in 269 patients at our institution. We examined total nucleated cell count, absolute counts, and percentages by WBC subtype. We compared CSF WBC values by tumor cell presence, patient vital status, tumor location, and the most common tumor types. Patients who died of their tumor had a lower CSF lymphocyte percentage and a higher absolute monocyte count in CSF at diagnosis. The presence of tumor cells in CSF was associated with fewer lymphocytes and monocytes. Ventricular tumors had higher CSF lymphocyte, monocyte, macrophage, and total nucleated cell counts than extraventricular tumors. Germ cell tumors, low-grade glioma, high-grade glioma, and ependymoma had lower macrophage counts or percentages compared to other tumor types. WBC profile in CSF at pediatric CNS tumor diagnosis correlates with patient prognosis and presence of metastatic cells, along with tumor type and other tumor characteristics like relationship to the ventricles. Prospective CSF profiling and study may be useful to future immunotherapy and other pediatric CNS tumor clinical trials.

The utility of flow cytometry for the detection of tumor cells in cerebrospinal fluid of patients with acute leukemia

Central nervous system infiltration by acute leukemia is a poor prognosis variable, and conventional cytology is the gold standard for its diagnosis; the technique is highly specific but not sensitive. To improve the diagnosis, flow cytometry has been used in different studies, showing greater sensitivity in the detection of leukemic cells. This study aimed to evaluate the presence of tumor cells by flowcytometry and conventional cytology, in cerebrospinal fluid from patients with acute leukemia as well as its relationship with clinical and biological parameters. In total, 156 CSF samples from 55 children with acute leukemia were studied. We found the following results: FCM-/CC- 131/156; FCM+/CC- 19/156; FCM-/CC+ 0; FCM+/CC+ 1/156; FC-/CC suspicious 1/156; and FCM+/CC suspicious 4/156. Patients with B-cell acute lymphoblastic leukemia and FCM+ showed a lower response to steroid-treatment, abnormal karyotype, neurological symptoms, and worse relapsefree survival. Patients with T-cell acute lymphoblastic leukemia and FCM+ demonstrated association with thrombocytopenia. In conclusion, flow cytometry has greater sensitivity for the detection of tumor infiltration in cerebrospinal fluid, a finding that correlates with prognostic parameters in patients with acute leukemia.

Abstract PD4-03: Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with breast cancer

Abstract Introduction. Breast cancers that metastasize to the central nervous system (BrM) and progress to leptomeningeal disease (LMD) are commonly HER2 amplified. Triple negative breast cancer (TNBC) also may also acquire HER2 amplification and/or copy number changes (HER2+) in BrM and LMD due to genetic heterogeneity. LMD has a dismal prognosis if untreated, but advances in anti-HER2 therapy have been highly effective for improving outcomes in patients with HER2+ LMD. Current methods for evaluating HER2 status in LMD are extremely limited. Cerebrospinal fluid (CSF) cytology is of limited sensitivity with poor cellular yield and preservation due to technical artifacts of preparation that severely limit evaluation of HER2 by FISH or IHC. We evaluated the ability of CNSide™ to selectively enrich CSF tumor cells (CSF-TCs) and assess HER2 gene amplification or copy number gain in breast cancer patients with LMD. Methods. CSF specimens from 77 unique breast cancer patients with suspected LMD were collected using CEE-Sure™ CSF collection tubes and analyzed using the CNSide assay, both produced by Biocept, Inc (San Diego CA). CSF tumor cells (CSF-TCs) stabilized at ambient temperature in CEE-Sure for shipping. In the CLIA laboratory CSF-TCs are labeled using a proprietary mouse anti-human antibody cocktail with secondary biotinylated anti-mouse Ig. All CSF cells are then captured in a streptavidin-coated micro-fluidic channel and fluorescently labeled with DAPI, streptavidin, keratin and CD45 to discriminate nucleated CSF-TCs that are SA+ and cytokeratin+, but CD45 negative. CSF-TCs are digitally imaged, localized and counted with specific X-Y coordinates in the channel that allow further characterization of the HER2 status of each cell using FISH probes for centromere 17 and HER2. Results. Of 77 unique patient CSF specimens, 74.0% (57/77) demonstrated the presence of metastatic tumor cells (defined as cells which were DAPI positive, streptavidin positive, and CD45 negative). HER2 cells ranged from fewer than 10 cells to tens of thousands of cells recovered from a single collection tube of CSF. Of the 57 specimens in which malignant cells were identified, 56.1% (32/57) demonstrated HER2 amplification by FISH (defined as HER2:CEP17 ≥ 2.0 or ≥ 6 HER2 signals) and 43.9% (25/57) did not demonstrate HER2 amplification by FISH. Ten (10) patients also had polysomy of chromosome 17 as well as other gene alterations. Conclusion: HER2 amplification is frequently found in breast cancer patients with LMD and can be reliably detected in tumor cells isolated from CSF. Evaluation of HER2 status in CSF-TCs allows treating physicians the opportunity to rapidly confirm the presence of this biomarker and select a more effective targeted therapy against HER2 in patients who present with life-threatening complications of breast cancer. Additional studies are needed to fully establish the clinical utility of CNSide when using various anti-HER2 therapies or therapy combinations to treat breast cancer patients with LMD. Citation Format: Michael Dugan, Deanna Fisher, Nathan Sweed, Barbara Blouw, Julie Mayer. Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD4-03.

Abstract P2-01-12: Detection of circulating tumor cells in cerebrospinal fluid for patients with suspected breast cancer leptomeningeal metastases: A prospective study

Abstract Background Breast cancer (BC) is the most frequent cause of leptomeningeal metastases (LM). LM diagnosis is confirmed by the detection of tumor cells in the cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, even with optimal CSF sample volume and time to the analysis, the sensitivity of this technique is low, demanding repeated samples. Here, we aimed to evaluate the value of circulating tumor cell (CTC) detection in CSF using the CellSearch® system for LM diagnosis. Materials and Methods This prospective, monocentric study included adult BC patients with suspected LM (clinical and/or radiological signs). CSF samples from 1-3 lumbar puncture(s) were analyzed: protein level, conventional cytology (60 drops), and CTC detection with the CellSearch® system (60 drops, first lumbar puncture only). Sensitivity (Se) and specificity (Sp) were calculated, using the results of the conventional cytology as the gold-standard. Results Forty-nine eligible patients were included (Jan 2017-Jan 2020): median age 51.8, 95.9% women, 20.4% HER2+ BC, 93.8% previously diagnosed with metastatic BC, 89.8% with clinical symptoms. Among them, 40 were evaluable (CTC detection failure: n=8, eligibility criteria failure: n=1). Median sample volume was 3.0 mL for conventional cytology samples (median time to analysis: 22min) and 3.3 mL for CTC samples. Of the 40 evaluable patients, 18 had a positive cytology (on CSF sample n=°1/n°2: n=16/n=2) and were therefore diagnosed with LM using the gold-standard method. Protein level was elevated in 88.2% of these patients, compared with 45.1% of patients with negative CSF cytology (p=0.005). CTCs were detected in these 18 patients (median 5824 CTCs, range 93-45052). CTCs were also detected in 5/22 patients with a negative cytology (median 2 CTCs, range 1-44). Among them, one patient (44 CTCs) was diagnosed with a cytologically-proven LM 9 months later, while there was no further argument for LM in the other 4 patients’ history (1-3 CTC), who died of the extra-cerebral disease after a median time of 5.2 months (range 0.9-25.9). The detection of at least one CTC in CSF was associated with a Se of 100.0% (IC95% 82.4-100) and a Spe of 77.3% (IC95% 64.3-90.3) for the diagnosis of LM. Considering the number of CTC as a quantitative value, we determined the cut-off maximizing the Youden index using the ROC analysis (93 CTC). The detection of at least 93 CTC in CSF was associated with a Se and a Spe of 100.0% for the diagnosis of LM (area under the curve [AUC]: 1.0). Conclusion CTCs were detected with the CellSearch® system in all patients diagnosed with a cytologically-proven LM, as well as in a few patients without a cytological confirmation of LM. The prognosis of these patients with CSF cytology-/CTCs+ needs to be further investigated in a larger cohort. Citation Format: Amélie Darlix, Stéphane Pouderoux, Simon Thezenas, Alexis Bievelez, William Jacot, Laure Cayrefourcq, Nicolas Menjot-de-Champfleur, Cristina Leaha, Catherine Alix-Panabières. Detection of circulating tumor cells in cerebrospinal fluid for patients with suspected breast cancer leptomeningeal metastases: A prospective study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-12.

Quantitative assessment of circulating tumor cells in cerebrospinal fluid as a clinical tool to predict survival in leptomeningeal metastases.

Circulating tumor cells in cerebrospinal fluid are a quantitative diagnostic tool for leptomeningeal metastases from solid tumors, but their prognostic significance is unclear. Our objective was to evaluate CSF-CTC quantification in predicting outcomes in LM. This is a single institution retrospective study of patients with solid tumors who underwent CSF-CTC quantification using the CellSearch® platform between 04/2016 and 06/2019. Information on neuroaxis imaging, CSF results, and survival was collected. LM was diagnosed by MRI and/or CSF cytology. Survival analyses were performed using multivariable Cox proportional hazards modeling, and CSF-CTC splits associated with survival were identified through recursive partitioning analysis. Out of 290 patients with CNS metastases, we identified a cohort of 101 patients with newly diagnosed LM. In this group, CSF-CTC count (median 200 CTCs/3ml) predicted survival continuously (HR = 1.005, 95% CI: 1.002-1.009, p = 0.0027), and the risk of mortality doubled (HR = 2.84, 95% CI: 1.45-5.56, p = 0.0023) at the optimal cutoff of ≥ 61 CSF-CTCs/3ml. Neuroimaging findings of LM (assessed by 3 independent neuroradiologists) were associated with a higher CSF-CTC count (median CSF-CTCs range 1.5-4 for patients without radiographic LM vs 200 for patients with radiographic LM, p < 0.001), but did not predict survival. Our data shows that CSF-CTCs quantification predicts survival in newly diagnosed LM, and outperforms neuroimaging. CSF-CTC analysis can be used as a prognostic tool in patients with LM and provides quantitative assessment of disease burden in the CNS compartment.

Diagnosis of Leptomeningeal Metastasis in Women With Breast Cancer Through Identification of Tumor Cells in Cerebrospinal Fluid Using the CNSide™ Assay

Diagnosis of LM is limited by low sensitivity of cerebrospinal fluid (CSF) cytopathology. Detecting tumor cells in CSF (CSF-TCs) might be more sensitive. We evaluated if CNSide (CNSide), a novel assay for tumor cell detection in CSF, can detect CSF-TCs better than conventional CSF cytology. We enrolled adults with metastatic breast cancer and clinical suspicion for LM to undergo lumbar puncture (LP) for CSF cytopathology and CNSide. CNSide captured CSF-TCs using a primary 10-antibody mixture, streptavidin-coated microfluidic channel, and biotinylated secondary antibodies. CSF-TCs were assessed for estrogen receptor (ER) expression by fluorescent antibody and HER2 amplification by fluorescent in situ hybridization (FISH). CSF cell-free DNA (cfDNA) was extracted for next-generation sequencing (NGS). Leptomeningeal disease was defined as positive CSF cytology and/or unequivocal MRI findings. We calculated sensitivity and specificity of CSF cytology and CNSide for the diagnosis of LM. Ten patients, median age 51 years (range, 37-64), underwent diagnostic LP with CSF evaluation by cytology and CNSide. CNSide had sensitivity of 100% (95% Confidence Interval [CI], 40%-100%) and specificity of 83% (95% CI, 36%-100%) for LM. Among these patients, concordance of ER and HER2 status between CSF-TCs and metastatic biopsy were 60% and 75%, respectively. NGS of CSF cfDNAidentified somatic mutations in three patients, including one with PIK3CA p.H1047L in blood and CSF. CNSide may be a viable platform to detect CSF-TCs, with potential use as a diagnostic tool for LM in patients with metastatic breast cancer. Additional, larger studies are warranted.

BIOM-05. CASE SERIES OF MULTI-INSTITUTIONAL UTILITY OF CNSide TO MANAGE LEPTOMENINGEAL DISEASE IN PATIENTS WITH METASTATIC BREAST CANCER

Abstract INTRODUCTION Leptomeningeal Disease (LMD) occurs in 5% of breast cancer patients. Diagnosing LMD remains challenging. Current standard of care has limited sensitivity and is inadequate for monitoring treatment response. Biocept’s CNSide™ is a proprietary assay utilizing a 10-antibody capture cocktail with microfluidic chamber that quantitatively detects tumor cells in the cerebrospinal fluid (CSF). We present a case series using CNSide to manage LMD of 4 unique breast cancer patients treated at three different institutions and demonstrate its impact on clinical management. METHODS Patients were treated at Smilow Cancer Hospital at Yale-New Haven (1 patient), Northwestern Medicine Lou and Jean Malnati Brain Tumor Institute (1 patient) and Barrow Neurological Institute (2 patients). All patients received intrathecal treatment (IT) via an Ommaya Reservoir. CSF tumor cells were detected via cytology and CNSide at diagnosis (3 patients) and throughout treatment (4 patients). RESULTS At diagnosis, CNSide detected tumor cells in 3/3 patients, vs 2/3 patients for cytology. The fourth patient was diagnosed with LMD before CNSide was available. CNSide detected CSF tumor cells in 9/9 (100%) of measurements, vs 4/9 (44%) for cytology for samples analyzed in parallel. Throughout treatment, CNSide was able to track the quantitative LMD response and showed a decrease in CSF tumor cells in all four patients, ranging from 99.7% (from 773 to 2 cells, 1 patient) to 100% (from 4447 to 0 cells; and from 33 to 0 cells, 2 patients). CONCLUSION Intrathecal treatment of LMD via Ommaya reservoir is not widely adopted across the US. Our experience suggests that using CNSide for quantitative CSF tumor cell detection may aid in diagnosing LMD, as well as in quantifying response to treatment particularly in the setting of intrathecal therapy. However, larger prospective clinical trials are needed to establish the role of CNSide in the diagnosis and management of LMD.

P14.04 Detection of circulating tumor cells in cerebrospinal fluid for patients with suspected breast cancer leptomeningeal metastases. A prospective study

Abstract BACKGROUND Breast cancer (BC) is the most frequent cause of leptomeningeal metastases (LM). LM diagnosis is confirmed by the detection of tumor cells in the cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, even with optimal CSF sample volume and time to the analysis, the sensitivity of this technique is low, demanding repeated samples. Here, we aimed to evaluate the value of circulating tumor cell (CTC) detection in CSF using the CellSearch® system for LM diagnosis. MATERIAL AND METHODS This prospective, monocentric study included adult BC patients with suspected LM (clinical and/or radiological signs). CSF samples from 1–3 lumbar puncture(s) were analyzed: protein level, conventional cytology (60 drops), and CTC detection with the CellSearch® system (60 drops, first lumbar puncture only). Sensitivity (Se) and specificity (Sp) were calculated, using the results of the conventional cytology as the gold-standard. RESULTS Forty-nine eligible patients were included (Jan 2017-Jan 2020): median age 51.8, 95.9% women, 20.4% HER2+ BC, 93.8% previously diagnosed with metastatic BC, 89.8% with clinical symptoms. Among them, 40 were evaluable (CTC detection failure: n=8, eligibility criteria failure: n=1). Median sample volume was 3.0 mL for conventional cytology samples (median time to analysis: 22min) and 3.3 mL for CTC samples. Of the 40 evaluable patients, 18 had a positive cytology (on CSF sample n=°1/n°2: n=16/n=2) and were therefore diagnosed with LM using the gold-standard method. Protein level was elevated in 88.2% of these patients, compared with 45.1% of patients with negative CSF cytology (p=0.005). CTCs were detected in these 18 patients (median 5824 CTCs, range 93-45052). CTCs were also detected in 5/22 patients with a negative cytology (median 2 CTCs, range 1–44). Among them, one patient (44 CTCs) was diagnosed with a cytologically-proven LM 9 months later, while there was no further argument for LM in the other 4 patients’ history (1–3 CTC), who died of the extra-cerebral disease after a median time of 5.2 months (range 0.9–25.9). The detection of at least one CTC in CSF was associated with a Se of 100.0% (IC95% 82.4–100) and a Spe of 77.3% (IC95% 64.3–90.3) for the diagnosis of LM. CONCLUSION CTCs were detected with the CellSearch® system in all patients diagnosed with a cytologically-proven LM, as well as in a few patients without a cytological confirmation of LM. The prognosis of these patients with CSF cytology-/CTCs+ needs to be further investigated in a larger cohort.

LMD-15. Beyond cytologY - A single institution experience using CNSideTM for diagnosing and monitoring treatment response in Non-Small Cell Lung Cancer patients with Leptomeningeal Carcinomatosis (LMC)

IntroductionLeptomeningeal Carcinomatosis (LMC) occurs in 3–9% of Non-Small Cell Lung Cancer (NSCLC) patients. Diagnosis of LMC includes clinical evaluation, imaging, and cytology. These have modest sensitivity and are inadequate for monitoring treatment response. Biocept’s CNSideTM is a proprietary assay utilizing a 10-antibody capture cocktail with microfluidic chamber that quantitatively detects tumor cells in the cerebrospinal fluid (CSF). Switch BlockerTM is a proprietary single gene assay that detects actionable mutations in the CSF. We describe a retrospective single institution experience using these assays in NSCLC patients with confirmed LMC or suspected LMC, treated between 2017 and 2021.MethodsFor fresh samples, CNSide and cytology were used to detect tumor cells, NGS and Switch Blocker was used to detect actionable mutations. Frozen samples were analyzed by NGS and/or Switch Blocker assays.ResultsCSF was collected from 30 samples (16 unique patients), of which frozen (8 unique patients) and fresh samples (8 unique patients; 5 with and 3 without LMC). CNSide detected tumor cells in 100% samples (10/10) vs cytology in 40% samples (4/10). Of those without LMC, neither CNSide nor cytology identified tumor cells. In patients with serial samples, CNSide tracked the clinical course. Analysis of frozen CSF by NGS identified mutations including EGFR in six (6), ALK in three (3) and BRAF in one (1) patient, which correlated with the primary tumor. The median survival from diagnosis of LMC for those with frozen samples was 71.6 weeks.ConclusionWe demonstrate that 1) survival of patients with LMC can be prolonged, especially when an actionable target is identified, 2) CNSide has greater sensitivity in detecting LMC than cytology, and 3) quantitative monitoring of CSF tumor cells can be used to guide initial and subsequent therapies. Larger clinical trials are needed to better establish the utility of CNSide in managing LMC.

Applicability and Prognostic Significance of Flow Cytometric Cerebrospinal Fluid Investigation in Children with Acute Lymphoblastic Leukemia

Conventionally central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is found by blasts detection in cerebrospinal fluid (CSF) via cytospin slides microscopy. It was shown earlier, that low levels of tumor cells, undetectable by cytomorphology (CM), could be found in several ALL cases using more sensitive techniques such as multicolor flow cytometry (MFC) (A. Popov et al, ASH-2011). Nevertheless prognostic value of these low levels of blasts in CSF is still unclear. The aim of present study was to evaluate the clinical significance of MFC application for initial diagnostics of leukemic cells in CSF of children with ALL.Patients and methods. Since December 2008 till November 2014 in 155 consecutive children older than 1 year with ALL initial CNS involvement was investigated by CSF flow cytometry. Study group included 65 girls and 90 boys aged from 1 to 16 years (median age 4 years) treated according to ALL MB-2008 protocol. 134 patients (86.5%) suffered from various types of B-cell precursor ALL (BCP-ALL), while remaining 21 cases (23.5%) were T-lineage ALL. According to the protocol stratification system 59 patients belonged to standard risk group (SRG), 87 - to intermediate risk group (ImRG) and 9 - to high risk group. CSF lesion was studied in parallel by microscopy of cytospin slides and by MFC. CNS status was classified as CNS1 (no blast cells in CSF), CNS2 (<5 WBC/ul CSF with blast cells), or CNS3 (≥5 WBC/ul CSF with blast cells, or other signs of CNS involvement). Antibodies' combinations for cytometric CSF assessment were developed on the basis of leukemic cells immunophenotype defined by initial diagnostic bone marrow investigation. Treatment outcome was estimated by cumulative incidence of relapse (CIR). Median of follow up was 5 years.Results. In 5 patients CNS3 status was defined. These patients were excluded from the further analysis due to obvious neuroleukemia signs. CNS2 was found in 23 of 150 remaining cases (15.3%) while 127 (84.7) had CNS1. Totally 53 out of 150 studied children (35.3%) had MFC-detectable CSF lesion. In 32 CNS1-patients leukemic blasts were found only by MFC. Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by MFC (median = 418, range 8-158171 and median = 34, range 5-2762 respectively, p<0.001). Thus MFC allows detecting tumor cells in CSF much more frequently (p<0.001) than conventional CM. This difference could be explained mainly by higher MFC sensitivity. Both CM and MFC presented results, valuable for outcome prediction. Children with CNS2 (n=23) had higher relapse incidence that patients with CNS1 (n=127): CIR 0.38(0.11) and 0.16(0.05) respectively, p=0.005. 53 MFC-positive patients displayed higher CIR compared to 97 MFC-negative ones: 0.26(0.06) and 0.15(0.05) respectively, p=0.019. Nevertheless MFC lacked prognostic impact in clinically relevant patients' groups. Among CNS1 cases the CIR difference between MFC-positive (n=32) and MFC-negative (n=95) groups was not significant: 0.19(0.07) and 0.15(0.05) respectively, p=0.148. SRG patients, who could be restratified according to the results of CSF inspection, also haven't displayed any difference in relapse incidence between 51 MFC-negative patients (CIR 0.18(0.07)) and 8 MFC-positive ones (CIR 0.27(0.17), p=0.313). In ImRG after exclusion of T-ALL cases cytometric CSF assessment also lacked its prognostic impact: MFC-negative group (n=33) had similar outcome to MFC-positive patients (n=33): CIR 0.08(0.06) and 0.17(0.07) respectively, p=0.239. In 37 initially MFC-positive cases follow-up CSF samples were also studied. Patients with fast clearance of CNS burden (n=24) displayed better outcome compared to children who remained MFC-positive (n=13), although these differences didn't achieve statistical significance: CIR 0.32(0.10) and 0.54(0.14) respectively, p=0.116.Conclusions. Our data shows that MFC is able to find in CSF even small number of leukemic blasts, which are undetectable by conventional cytospin slides microscopy. Despite of this advantage of cytometric CSF inspection, the prognostic value of MFC data remains controversial. In contrast, larger tumor cells populations that could be detected in CSF also by cytomorpology are more strictly associated with high incidence of relapse. Nevertheless this data should be confirmed on larger patients' cohort in multicenter trial. DisclosuresNo relevant conflicts of interest to declare.