Detection of Neoplasms by Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid

Abstract

Importance: Cerebrospinal fluid (CSF) cytologic testing and flow cytometry are insensitive for diagnosing neoplasms of the central nervous system (CNS). Such clinical phenotypes can mimic infectious and autoimmune causes of meningoencephalitis.

Objective: To ascertain whether CSF metagenomic next-generation sequencing (mNGS) can identify aneuploidy, a hallmark of malignant neoplasms, in difficult-to-diagnose cases of CNS malignant neoplasm.

Design, setting, and participants: Two case-control studies were performed at the University of California, San Francisco (UCSF). The first study used CSF specimens collected at the UCSF Clinical Laboratories between July 1, 2017, and December 31, 2019, and evaluated test performance in specimens from patients with a CNS malignant neoplasm (positive controls) or without (negative controls). The results were compared with those from CSF cytologic testing and/or flow cytometry. The second study evaluated patients who were enrolled in an ongoing prospective study between April 1, 2014, and July 31, 2019, with presentations that were suggestive of neuroinflammatory disease but who were ultimately diagnosed with a CNS malignant neoplasm. Cases of individuals whose tumors could have been detected earlier without additional invasive testing are discussed.

Main outcomes and measures: The primary outcome measures were the sensitivity and specificity of aneuploidy detection by CSF mNGS. Secondary subset analyses included a comparison of CSF and tumor tissue chromosomal abnormalities and the identification of neuroimaging characteristics that were associated with test performance.

Results: Across both studies, 130 participants were included (median [interquartile range] age, 57.5 [43.3-68.0] years; 72 men [55.4%]). The test performance study used 125 residual laboratory CSF specimens from 47 patients with a CNS malignant neoplasm and 56 patients with other neurological diseases. The neuroinflammatory disease study enrolled 12 patients and 17 matched control participants. The sensitivity of the CSF mNGS assay was 75% (95% CI, 63%-85%), and the specificity was 100% (95% CI, 96%-100%). Aneuploidy was detected in 64% (95% CI, 41%-83%) of the patients in the test performance study with nondiagnostic cytologic testing and/or flow cytometry, and in 55% (95% CI, 23%-83%) of patients in the neuroinflammatory disease study who were ultimately diagnosed with a CNS malignant neoplasm. Of the patients in whom aneuploidy was detected, 38 (90.5%) had multiple copy number variations with tumor fractions ranging from 31% to 49%.

Conclusions and relevance: This case-control study showed that CSF mNGS, which has low specimen volume requirements, does not require the preservation of cell integrity, and was orginally developed to diagnose neurologic infections, can also detect genetic evidence of a CNS malignant neoplasm in patients in whom CSF cytologic testing and/or flow cytometry yielded negative results with a low risk of false-positive results.

Figure 1.. Schematic of the Metagenomic Next-Generation Sequencing (mNGS) Test and Its Overall Performance in 2 Studies

A, Brain lesions release pathogen or cancer cell–free DNA into the cerebrospinal fluid (CSF). An mNGS test performed on all specimens assessed for aneuploidy and pathogens. B, CSF specimens were obtained after flow cytometry and/or cytologic testing. Of these, 55 were positive controls (ie, from patients with central nervous system [CNS] malignant neoplasm) and 70 were negative controls from patients with an ultimate diagnosis of infectious or autoimmune disease. Tumor fractions are based on the amplitude of the copy number change. Cerebrospinal fluid mNGS was 64% sensitive and 100% specific for detecting the subset of malignant neoplasms not detected by flow cytometry and/or cytologic testing. C, Cases and control patients were from the idiopathic neuroinflammatory disease study. Cerebrospinal fluid mNGS had 55% sensitivity and 100% specificity for CNS malignant neoplasm detection. CNV indicates copy number variations; NI, not interpretable.

Figure 2.. Undiagnosed Primary CNS Lymphoma Cases

A, Conventional methods of brain biopsy and cytologic testing of the cerebrospinal fluid (CSF) were nondiagnostic in case 44. Flow cytometry showed atypical cells and was not definitive. Diagnosis was confirmed by autopsy 2 weeks after the CSF sampling. B, Arrowheads highlight large, perivascular B cells that have large, irregular nuclei with loose chromatin; hematoxylin-eosin (H&E) stain was used, with original magnification ×200 (left image) and ×1000 (right image). C, The large cells were stained positive (brown) for Epstein-Barr encoding region (EBER) in situ hybridization as evidence of Epstein-Barr virus (EBV) RNA expression in malignant lymphoma. D and E, Copy ratio plots are from 2 patients enrolled in the neuroinflammatory disease case-control study.

Figure 3.. Other Undiagnosed Cases

For cases 129 and 134, the comparable copy ratio plots derived from directly sequencing the neoplasm are shown.

Figure 4.. Images of Abnormalities in 4 Cases With Presentations Suggestive of Neuroinflammatory Disease at or Near the Time of Brain Biopsy

A, In case 128, postcontrast T1-weighted images demonstrate thick areas of enhancement along nearly all ependymal surfaces, including the lateral ventricles (top image), third ventricle (bottom image), and fourth ventricle, resulting in acute hydrocephalus. The imaging appearance was believed to be consistent with disseminated tuberculosis and less likely to be lymphoma because of limited restricted diffusion. The final diagnosis was primary central nervous system (CNS) lymphoma. B, In case 129, postcontrast T1-weighted images demonstrate extensive, thin ependymal enhancement along the lateral ventricles (top image) and fourth ventricle (bottom image), with minimal adjacent signal abnormality in the parenchyma. The imaging appearance was believed to be most consistent with infectious ventriculitis and less likely to be carcinomatosis. The final diagnosis was melanoma. C, In case 138, motion-degraded T2-weighted fluid attenuated inversion recovery (T2/FLAIR; top image) image and apparent diffusion coefficient (bottom image) demonstrate asymmetric white matter signal abnormality with variable foci of mildly restricted diffusion. In this immunocompromised patient, images were believed to be potentially consistent with progressive multifocal leukoencephalopathy. The final diagnosis was intravascular lymphoma. D, In case 130, postcontrast T1-weighted (top image) and T2/FLAIR (bottom image) sequences demonstrate expansile signal abnormality with areas of heterogeneous enhancement. Glioblastoma was diagnosed using brain biopsy. Arrowheads indicate areas of abnormalities.