Diagnosis of leptomeningeal metastasis in women with breast cancer through identification of tumor cells in cerebrospinal fluid using the CNSide™ assay

Micro Abstract

Leptomeningeal metastasis (LM) is a devastating complication of metastatic breast cancer; however, diagnosis is limited by low sensitivity of cerebrospinal fluid cytopathology. This study evaluates a novel assay, CNSide™, and demonstrates 100% sensitivity for LM and additionally, capacity for receptor assessment and mutation identification. CNSide™ may be a viable platform to detect LM, with potential use as a diagnostic tool.

Introduction

Leptomeningeal metastasis (LM) is a late-stage complication of systemic malignancies in which malignant cells seed the meninges and result in metastatic involvement of the brain parenchyma, spinal cord, cranial and/or peripheral nerves.¹ Despite advances in systemic and radiation therapies, LM is associated with a poor prognosis, with median overall survival less than one year.^2–4 The reported incidence of LM among patients with advanced malignancies is as high as 8%, and the most common primary tumor associated with LM is breast cancer.^5,6 The incidence of LM is thought to be increasing because of improvement in imaging modalities, longer survival among patients with metastatic malignancies, and poor CNS penetration of many systemic therapies. Recent clinical trials have evaluated the role for intravenous and intrathecal chemotherapy with whole brain radiation therapy;^7–9 however, there is currently no consensus regarding the treatment of LM.^10,11

Diagnosis of LM has traditionally been based on cytopathologic analysis of cerebrospinal fluid (CSF), with the presence of malignant cells in CSF considered the gold standard. However, CSF cytopathologic analysis has low sensitivity, approximately 50–75% on the first lumbar puncture, and is highly examiner-dependent.^12–17 Repeat, multi-site and high-volume lumbar punctures are often required, which may increase sensitivity up to 80–90%, but are associated with potential complications, treatment delays, and patient discomfort.^13,18 While brain and spinal magnetic resonance imaging (MRI) has been incorporated into the initial diagnostic evaluation for LM, MRI findings can be equivocal, and unequivocal findings may only appear in late-stage disease.^6,19 There is therefore an unmet need for a sensitive diagnostic tool for LM that can allow for accurate diagnosis without repeated lumbar punctures.

Novel methods to quantify and characterize circulating tumor cells (CTCs) in blood have been evaluated in CSF samples as potential diagnostic modalities for LM. Although isolated via similar methods, tumor cells in the CSF (CSF-TCs) do not directly communicate with circulating blood, and hence are considered confirmatory of metastatic involvement of the central nervous system rather than just incidental CTCs in peripheral circulation. The CNSide™ (CNSide) assay for CSF is based on a Cell Enrichment and Extraction collection tube (CEE-Sure™), microfluidic platform and antibody capture method (Biocept, San Diego, CA), that was originally designed to capture and detect CTCs in the peripheral blood. The capture of these tumor cells has been developed to be independent of expression of any single epithelial surface markers such as the epithelial cell adhesion molecule (EpCAM). CNSide, like the Target Selector™ (TS) for blood, uses a biotin-tagged antibody cocktail against a variety of antigens, including both EpCAM and non-EpCAM epithelial and mesenchymal antigens that was originally developed to more efficiently detect CTCs in the blood than assays utilizing antibodies against EpCAM alone.^20–23 CNSide and TS also allow for more detailed phenotypic and genomic characterization of tumor cells through use of immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH).^21–24

In this study, we evaluated whether CNSide can detect CSF-TCs better than conventional CSF cytology to diagnose LM among women with metastatic breast cancer (MBC), with the hypothesis that CNSide can detect CSF-TCs in the CSF and is a sensitive tool for the diagnosis of LM. In additional analyses, we performed next-generation sequencing (NGS) of cell-free DNA (cfDNA) in the CSF with comparison to NGS on peripheral blood CTCs, and assessed the feasibility of determining hormone receptor and HER2 status on CSF-TCs using the CNSide and TS technology.

Patients and Methods

Study Cohort

We conducted a prospective study among patients with advanced or metastatic breast cancer and clinically suspected or confirmed leptomeningeal disease at Columbia University Irving Medical Center (CUIMC) in New York, NY. Patients aged 18 years or older with a histologically proven metastatic solid tumor or primary central nervous system (CNS) malignancy were eligible for enrollment if they were planned for a diagnostic lumbar puncture (LP) for clinical suspicion of LM and/or unequivocal or suspicious findings for LM on magnetic resonance imaging (MRI). After study enrollment and evaluation, patients were considered to have a definitive diagnosis of LM if they had malignant cells detected by CSF cytopathologic analysis (positive cytology) and/or unequivocal MRI findings. Unequivocal MRI findings were defined as leptomeningeal enhancement with subarachnoid nodules, enhancement in basal cisterns, or enhancement/clumping of nerve roots. Findings such as multiple superficial brain metastases, intraventricular masses, dural enhancement associated with epidural metastasis, or new hydrocephalus was considered suspicious but non-diagnostic. There were no limitations on the number of systemic therapies received prior to enrollment. Patients were recruited from the CUIMC medical oncology and neuro-oncology clinics. The study was approved by the CUIMC Institutional Review Board (AAAQ4761) and written informed consent was obtained from each participant.

For enrolled patients, we collected clinicopathologic data including patient age, sex, primary tumor histology, and prior systemic therapies. We also reviewed the electronic health record and collected information about the most recent biopsy of a metastatic site for estrogen receptor (ER), progesterone receptor (PR), and HER2 status.

CSF and Peripheral Blood Collection

Study participants underwent LP and standard CSF assessment at CUIMC consisting of intracranial pressure measurement, CSF protein, glucose, white and red cell analysis, infectious cultures, as well as conventional cytopathology analysis (cytocentrifuge). In addition to standard CSF collection, 10 mL CSF sample was obtained in CEE-Sure™ collection tubes (Biocept Inc., San Diego, CA) for CSF-TC assessment, along with two 10 mL vials of peripheral blood for evaluation of serum CTCs and cfDNA in CEE-Sure™ collection tubes. Samples were delivered to Biocept’s laboratory for processing and evaluated for CSF-TCs using CNSide microchannel technology.

CSF and Peripheral Blood Analysis

All samples were collected in CEE-Sure™ collection tubes. CSF samples were spun down and approximately 1mL containing the cell pellet was processed for CSF-TC testing and the supernatant used for molecular testing. Following centrifugation of whole blood, plasma was removed for molecular testing and the buffy coat layer was isolated for CTC testing using a percoll density gradient. CSF-TC testing was performed by incubating with a primary 10-antibody mixture that targets cell surface epitopes, followed by incubation with biotinylated secondary antibody. The primary capture antibody mixture contained anti-EpCAM, tumor-associated calcium signal transducer 2, anti-c-MET, anti-Folate-binding protein receptor, anti-N-Cadherin, anti-CD318, and anti-mesenchymal stem cell antigen. Labeled CSF-TCs are then captured in a streptavidin coated microfluidic channel and stained with a mixture of anti-cytokeratin 17, 18, 19, pan-cytokeratin, CD45, streptavidin and DAPI. CSF-TC enumeration analysis was undertaken and classified either as cytokeratin positive (CK+/CD45-/DAPI+) or cytokeratin negative (SA+/CD45-/DAPI+). CSF-TCs were assessed for ER expression by fluorescent antibody and for HER2 amplification evaluated by FISH. The supernatant from CSF and plasma from peripheral blood was used to extract cell free DNA and RNA. Amplicon-based NGS was performed using the 12 gene TS Breast NGS Panel powered by Oncomine ThermoFisher. Libraries were prepared, templated on the Ion Chef and sequenced using the Ion S5 XL system. Data analysis was performed using the Ion Reporter software (version 5.10).

Data Analysis

The study was designed to enroll 36 evaluable patients to have 80% power to detect a 25% improvement in the sensitivity to detect leptomeningeal metastasis using CSF-TCs by CNSide vs. standard cytopathologic analysis on the first lumbar puncture (75% vs. 50%) with a two-sided test at a significance level of 0.05. The sample size justification assumed that 90% of patients (N=32) would have LM. However, given low accrual, the study was ultimately stopped early. Thus, the analyses are descriptive in nature.

Sensitivity and specificity of CNSide and cytopathology for LM was calculated among patients with CSF samples evaluable for cytopathologic and CNSide analyses and are reported with respective 95% exact binomial confidence intervals. Patient baseline characteristics were summarized and reported using median and range for continuous variables and frequencies (percent) for categorical variables. We evaluated characteristics of CSF-TCs and cfDNA collected from CSF and compared results to CTCs and cfDNA assays collected simultaneously from peripheral blood. These results were reported by patient and summarized among patients. In addition, we assessed and reported concordance of the primary and/or metastatic tumor with receptor ER and HER2 on CSF-TCs using CNSide technology.

Results

Between January 2017 and December 2019, ten women with MBC and clinical or radiologic suspicion for LM disease were enrolled and underwent a lumbar puncture (Table 1). Median age at enrollment was 51 years (range, 37–64). Patients had received a median of 3 (range, 0 – 8) prior lines of systemic therapy in the advanced/metastatic setting. While all patients had at least clinical concern for leptomeningeal metastases, only four (40.0%) had definitive LM by either CSF cytology only (n = 3) or MRI findings only (n = 1).

Table 1.

Baseline clinicopathologic characteristics of enrolled patients, n = 10.

Characteristic	No. of patients (%)
Median age, in years (range)	51 (37 – 64)
Female sex	10 (100%)
Median number of prior lines of systemic therapy for advanced/metastatic disease (range)	3 (0 – 8)
Definitive leptomeningeal disease	4 (80.0%)
CSF cytology	3 (30.0%)
MRI	1 (10.0%)
CSF evaluable for cytology	9 (90.0%)

The results of CSF analysis by cytopathology and CNSide are summarized in Table 2. All patients had evaluable CSF samples for CNSide, while nine patients had evaluable CSF for cytopathology. Among the four patients with definitive LM, all four (100%) had CSF-TCs detected by CNSide, while three had CSF cytology positive for malignant cells and one patient did not have evaluable CSF for cytology. The sensitivity and specificity of CNSide for LM were 100% (95% Confidence Interval [CI], 40–100%) and 83% (95% CI, 36–100%), respectively. The sensitivity and specificity of cytopathology for LM were 100% (95% CI, 29–100%) and 100% (95% CI, 54–100%), respectively. Five patients had CSF-TCs detected in CSF samples by CNSide, of whom three (60%) had positive CSF cytology, one had negative cytology, and one patient did not have evaluable CSF cytology. Of note, one patient with CSF-TCs detected by CNSide did not carry a definitive diagnosis of LM by either cytopathology or MRI.

Table 2.

Patient-level results of cerebrospinal fluid analysis by standard cytopathologic analysis and CNSide™, and phenotypic and genomic characterization of CSF-TCs using CNSide™, n=10.

ID	CSF cytology at CNSide collection	CNSide CSF-TCs	Definitive LM	Receptor status on metastatic biopsy (ER/HER2)	CNSide CSF receptor status (ER/HER2)	ER Status Concordant with CNSide?	HER2 Status Concordant with CNSide?	Genomic Alteration in CSF	Genomic Alteration in blood (CNSide)
1	+	+	Y	−/−	+/−	N	Y	None detected	None detected
2	Not performed	+	Y	−/−	−/+	Y	N	TP53 p.K132R	None detected
3	−	−	N	+/−	N/A	N/A	N/A	None detected	ESR1 p.E380Q ESR1 p.Y537D TP53 p.R273H
4	−	−	N	unk	N/A	N/A	N/A	None detected	None
5	+	+	Y	−/−	−/−	Y	Y	None detected	TP53 p.V272M TP53 pC238Y
6	+	+	Y	+/−	−/−	N	Y	PIK3CAp.H1047L	PIK3CA p.H1047L
7	−	−	N	unk	N/A	N/A	N/A	None detected	None detected
8	−	−	N	−/−	N/A	N/A	N/A	Not performed	Not performed
9	−	−	N	unk	N/A	N/A	N/A	CCND1 deletion	PIK3CA p.E542K PIK3CA p.E545K PIK3CA p.E726K
10	−	+	N	−/−	−/unk	Y	N/A	None detected	None detected

CNSide = CNSide™, CSF = cerebrospinal fluid, LM = leptomeningeal disease, ER = estrogen receptor

Y = yes, N = no, + = positive for malignant cells or CSF-TCs; − = negative for malignant cells or CSF-TCs

Unk = unknown, N/A = not available

The results of receptor status on CSF-TCs detected by CNSide, and concordance of receptor status between metastatic biopsy and CSF-TCs, are also presented in Table 2. Concordance of receptor status between metastatic biopsy and CSF-TCs was also assessed. Among the five patients with CSF-TCs detected, ER status was concordant with metastatic tumor tissue in three (60.0%). HER2 status was not able to be assessed in one patient, but was found to be concordant in three of the four evaluable patients (75.0%). Of note, a repeat metastatic biopsy was not required as part of this study; however, most metastatic biopsies used for comparison to CSF-TCs were performed within one year of CSF analysis for this study.

Patients also underwent analysis for genomic alterations in cfDNA in the CSF and plasma (Table 2). Eight patients had evaluable cfDNA from the CSF for detection of genomic alterations, and analysis identified genomic alterations in cfDNA from three patients, with alterations detected in TP53, PIK3CA, and CCND1. Of note, the patient with CCND1 deletion detected in the CSF had negative CSF cytology and no CSF-TCs were detected. Analysis of the cfDNA from plasma in nine patients identified genomic alterations in four, with multiple alterations noted in three patients. Affected genes included ESR1, TP53, and PIK3CA. Notably, a concordant PIK3CAp.H1047L mutation were detected in the CSF and serum of one patient. The remaining patients with alterations noted in the CSF and/or serum did not have concordance.

Discussion

Our findings support that CNSide may be a viable platform for the detection of CSF-TCs in the CSF, with potential use as a sensitive diagnostic tool for LM. Among ten patients with MBC and a clinical and/or radiologic suspicion of LM, CNSide analysis had a sensitivity of 100% (95% CI, 40–100%) and specificity of 83% (95% CI, 36–100%) for LM. In addition, ER and HER2 status were concordant between CSF-TCs and primary or metastatic tissue in 60 and 75 percent, respectively. CNSide was also able to identify genomic alterations in genes including PIK3CA in the CSF cfDNA.

Our findings are in agreement with recent data that an EpCAM-based assay had a sensitivity of 94% and specificity of 100% for LM among patients with metastatic epithelial malignancies, compared with a sensitivity of 76% for standard cytopathologic analysis.²⁵ Epithelial cell adhesion molecule (EpCAM) is a surface antigen on epithelial malignancies including breast cancers, and EpCAM assays such as CellSearch have been developed to detect tumor cells in the CSF with reported sensitivities of 76%–100%.^{14–17,25–27} However, the use of EpCAM assays is limited to metastatic epithelial malignances, and also carries a theoretical risk of false-positive results from CSF contamination with skin epithelial cells during lumbar puncture.^25,27 In addition, loss of epithelial markers can occur during epithelial-mesenchymal transition, which is thought to be a key step in the development of metastasis and could result in decreased detection of tumor cells.²⁰ The potential advantage of CNSide over EpCAM-based assays is its incorporation of non-epithelial antigens, which could expand its application to non-epithelial malignancies. Future larger, prospective studies are therefore warranted to further compare the performance of CNSide to cytopathologic analysis in the diagnosis of LM, including among patients with non-epithelial malignancies.

We also evaluated whether ER expression and HER2 status could be determined from CSF-TCs. We found that ER and HER2 status were concordant between CSF-TCs and metastatic biopsy in 60 and 75 percent of patients with CSF-TCs detected, respectively. Among those with discordant ER and HER2 status, possible explanations include that the metastatic biopsy used for comparison was not consistently performed at the time of CSF sampling and also that receptor conversion due to clonal evolution might have occurred during the course of metastatic disease. Receptor conversion for hormone receptors and HER2 occurs frequently in the course of disease progression in breast cancer, particularly in the CNS;²⁸ approximately 36% of patients with MBC to the CNS have discordance in receptor status between brain metastases and primary tumor, with the majority of this subset also showing discordance between brain and extracranial metastases.²⁹ We demonstrated that ER and HER2 status could be determined from CSF-TCs collected using CNSide with high concordance with other metastatic sites, similar to our previous findings using TS to detect CTCs in the peripheral blood of patients with MBC,²⁴ and supporting further evaluation of its potential application in patients with MBC and confirmed or suspected LM. Characterization of hormone receptor and HER2 status in the CSF in these patients could inform treatment decisions. For example, in a patient with MBC confined to the leptomeninges without another site accessible to biopsy, the ability to evaluate these markers would be critical to guide choice of systemic therapies. Confirmation of HER2 status on CSF-TCs in particular could have treatment implications, with consideration of therapies with better CNS penetration such as tucatinib³⁰ in patients with confirmed HER2-positive cancer cells in the CSF.

Lastly, we also demonstrated that NGS of cfDNA in the CSF using CNSide can identify potentially actionable mutations among patients with MBC, including one patient with the same PIK3CA sequencing variant identified in both CSF and blood. Given that the identification of variants in PIK3CA and other genes can have treatment implications in MBC,³¹ CNSide could have clinical utility among patients with CNS metastatic disease. Similarly, previous studies using EpCAM-based assays and NGS have identified actionable mutations in the CSF of patients with metastatic malignancies, including EGFR mutations in non-small cell lung cancer.^15,25 Further studies using CNSide should evaluate its ability to detect genomic variants in the CSF of patients with non-breast malignancies.

Limitations of our study include its small number of enrolled patients at a single academic institution. During enrollment, there was a dedicated neuro-oncology coordinator focused on accrual to this study. There were regular weekly meetings in neuro-oncology, as well as breast oncology, to discuss barriers to accrual. The low accrual number may be due to the fact that, despite this, not all patients with potential central nervous system disease were referred to neuro-oncology for a consultation. The number of samples being collected during the study was not thought to be a barrier; rather, the number of potential patients being seen in neuro-oncology clinic. The small number of patients might limit its generalizability as well as statistical comparison of the sensitivity and specificity of CNSide to standard cytopathologic analysis for LM. In particular, sensitivity and specificity of CNSide for LM had wide confidence intervals, and therefore require further evaluation in a larger patient cohort. Our cohort also only included patients with metastatic breast cancer, and so requires additional investigation in patients with other cancers. In order to address these limitations, a larger, multicenter study is necessary to allow evaluation of CNSide in larger, more diverse patient cohorts, including patients with other malignancies, to improve the generalizability of our findings. To operationalize this study, having a system arranged in which all patients with breast cancer and known or suspected central nervous system disease are seen in neuro-oncology will help ensure an adequate sample size.

Conclusion

CNSide may be a viable platform for the detection of tumor cells in the CSF with high sensitivity for LM among women with metastatic breast cancer, and allows for phenotypic and genomic characterization of CTCs in peripheral blood and cfDNA in the CSF. Future larger, multicenter prospective studies are warranted to further investigate CNSide as a diagnostic tool for LM among patients with metastatic breast cancer.

Clinical Practice Points.

• LM is a devastating complication of metastatic breast cancer with overall survival of less than one year. The current gold standard for diagnosis of LM is based on cytopathologic analysis of CSF; however, cytopathologic analysis has low sensitivity and is highly examiner dependent.

• This study found that CNSide™, a novel technology, has sensitivity of 100% and specificity of 83% for detecting LM using tumor cells from CSF samples of patients with metastatic breast cancer. CNSide™ was also able to determine ER and HER2 receptor status with concordance between CSF tumor cells and metastatic biopsy of 60% and 75%, respectively.

• CNSide™ may have treatment implications as it can be used to perform NGS of cfDNA in the CSF to identify genomic variants with potentially actionable mutations in patients with metastatic breast cancer.