Real-world experience with circulating tumor DNA in cerebrospinal fluid from patients with central nervous system tumors

Abstract

The characterization of genetic alterations in tumor samples has become standard practice for many human cancers to achieve more precise disease classification and guide the selection of targeted therapies. Cerebrospinal fluid (CSF) can serve as a source of tumor DNA in patients with central nervous system (CNS) cancer. We performed comprehensive profiling of CSF circulating tumor DNA (ctDNA) in 711 patients using an FDA-authorized platform (MSK-IMPACT™) in a hospital laboratory. We identified genetic alterations in 489/922 (53.0%) CSF samples with clinically documented CNS tumors. None of 85 CSF samples from patients without CNS tumors had detectable ctDNA. The distribution of clinically actionable somatic alterations was consistent with tumor-type specific alterations across the AACR GENIE cohort. Repeated CSF ctDNA examinations from the same patients identified clonal evolution and emergence of resistance mechanisms. ctDNA detection was associated with shortened overall survival following CSF collection. Next-generation sequencing of CSF, collected through a minimally invasive lumbar puncture in a routine hospital setting, provides clinically actionable cancer genotype information in a large fraction of patients with CNS tumors.

Fig. 1

Genomic alterations detected in CSF-ctDNA. a Oncoplot of the most frequently altered genes, stratified by broad tumor categories. Each column represents an individual sample. Plot includes non-synonymous mutations, indels, copy number alterations and structural variants. Upper bars depict TMB levels; the dashed green line indicates a TMB of 10 muts/Mb. Lowest track indicates the tumor category. Multi_Hit refers to those genes that were mutated more than once in the same sample. b, c Circos plot of the 186 structural variants identified. Arrows highlight select recurrent alterations with the most clinical relevance, further stratified by number and clinical implication as diagnostic or therapeutic. d Distribution of observed mutation rates across CSF samples sequenced; a threshold of 13.8 mutations/Mb was considered indicative of high mutation burden based on historical analysis of 10,000 tumor samples by MSK-IMPACT testing (left). Dominant mutation signatures identified in cases with high mutation burden. The percent of cases harboring a dominant mutation signature is shown for each broad tumor category (right panel). MMR: Mismatch repair deficiency; UV: Ultraviolet light; TMZ: Temozolomide

Fig. 2

Clinical Relevance of CSF-ctDNA positivity. a Alterations detected in cfDNA from CSF were annotated and stratified by their level of clinical actionability according to the OncoKB precision oncology knowledge base. The proportions were compared to the AACR-Genie MSK cohort of solid tumor (n = 47,271). There is a relative enrichment for level 1 alterations due to the use of the assay for monitoring of patients on targeted therapies. b Survival curves showing that detection of ctDNA in CSF is associated with lower survival probability

Fig. 3

CSF sampling in NSCLC. a 77 samples with actionable driver alterations detected in CSF were compared to results from prior tissue biopsies. Driver alterations initially detected in the tumor tissue were universally detected in the CSF. Each column represents 1 patient. Blue boxes designate those samples where both CSF and tissue sequencing demonstrated the same driver alteration. In two cases, the mutation detected in the CSF was distinct form the one detected in the tumor. In both cases, retrospective review demonstrated the presence of multifocal lung disease with the metastasis representing a separate primary that was not previously sequenced. b Among patients with EGFR sensitizing mutations, sequencing of CSF from 28 patients detected several additional alterations associated with secondary resistance, including mutations in EGFR, and alterations in other genes (MET, PIK3CA, BRAF). c Representative case of a patient with EGFR mutated lung adenocarcinoma and monitoring starting at the time of suspected CNS metastasis. 7 CSF samples are obtained demonstrating the gradual emergence of several resistance mechanisms associated with treatment with EGFR inhibitors (T790M, L718V and L718Q. The table displays the mutations detected in each sample sequenced, along with the corresponding VAF’s (%), highlighted according to the color scale (bottom left). Lowest track denotes the classification of the EGFR mutations as sensitizing (L1, green) or associated with acquired resistance (R1 standard care resistance; R2 investigational resistance, red) according to OncoKB. L4 (dark gray) denotes an alteration with compelling biological evidence that supports the biomarker as being predictive of response to a drug. d Survival curves for NSCLC patients demonstrate that detection of ctDNA in CSF is associated with lower survival probability

Fig. 4

Pre-analytic factors associated with CSF-ctDNA positivity. a Stratification of samples based on disease type (primary CNS tumor vs metastasis) shows that that metastatic tumors have higher rates of ctDNA positivity than primary tumors. b Samples are stratified by the volume of CSF received for testing. While genetic alterations could be detected even in the context of very low volume samples, the rate of positivity was critically impacted for those samples below 2 ml. These samples were associated with rates of ctDNA positivity between 8 and 20%. The rate of positivity increases as volumes reach 5 ml and above. Yellow bars indicate the proportion of samples that are ctDNA positive. Blue bars indicate those that are ctDNA negative (no detected genetic alterations). c Analysis of rates of positivity for samples according to time to extraction. Across the entire cohort, increased time to extraction was not associated with increased proportion of ctDNA negative samples. Samples extracted outside the stability criteria of STREK tubes (> 14 days) constituted a very small proportion of the samples–this very small subset demonstrated a drop in the rate of positivity compared to those extracted before 14 days but the number was too low for a conclusive analysis. d, e Comparisons of total DNA yields and sequencing coverages between ctDNA + and ctDNA- samples. Overall, the proportion of ctDNA + samples increased with higher DNA yield and, consequently, higher sample coverages. f A broad range of coverages are found across CSF samples. Top graph shows the range of coverages across the entire cohort. Lower panel and insert (right) display the zoomed views of the samples with lowest coverages. Despite the low coverages, detection of genetic alterations remains possible in many cases below 50× due to the high proportion of ctDNA in CSF samples (not diluted by cfDNA from hematopoietic components). Higher proportion of samples have ctDNA detected when sample coverage increases