Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for primary central nervous system lymphoma

Abstract

Background: Primary central nervous system lymphomas (PCNSLs) are highly aggressive tumors. Chemotherapy has improved prognosis significantly; however, early diagnosis is crucial for effective treatment. Presently, the diagnosis of PCNSL depends on histopathology of tumor biopsies. We have previously demonstrated differential expression of microRNAs in cerebrospinal fluid (CSF) samples from patients with PCNSL. Based on promising findings about circulating U2 small nuclear RNA fragments (RNU2-1f) as novel blood-based biomarkers for pancreatic, colorectal, and lung cancer, we investigated RNU2-1f in the CSF of PCNSL patients.

Methods: CSF was collected from patients with PCNSL (n = 72) and control patients with various neurologic disorders (n = 47). Sequential CSF samples were collected from 9 PCNSL patients. RNU2-1f levels were measured by real-time polymerase chain reaction.

Results: Measurement of RNU2-1f levels in CSF enabled the differentiation of patients with PCNSL from controls with an area under the curve (AUC) of 0.909 with a sensitivity of 68.1% and a specificity of 91.4%. The diagnostic accuracy was further improved by combined determination of RNU2-1f and miR-21, resulting in AUC of 0.987 with a sensitivity of 91.7% and a specificity of 95.7%. In consecutive measurements of RNU2-1f, which were performed in 9 patients at different stages of the disease course, RNU2-1f CSF levels paralleled the course of the disease.

Conclusions: Our data suggest that the measurement of RNU2-1f detected in CSF can be used as a diagnostic marker and also as a possible marker for treatment monitoring. These promising results need to be evaluated within a larger patient cohort.

Keywords: cerebrospinal fluid (CSF); disease course; primary central nervous system lymphoma; small nuclear RNA (snRNA).

Fig. 1.

Stability of endogenous RNU2-1f in cerebrospinal fluid (CSF) samples. To evaluate the thermostability, RNU2-1f was quantified in CSF samples collected from a patient with primary central nervous system lymphoma (PCNSL) and from a healthy control and stored at room temperature (22°C) for a maximum of 3 days. At times indicated, aliquots were removed and assayed for RNU2-1f by qRT-PCR. Data points were calculated by subtracting cycle threshold (Ct)-values (Ct(RNU2-1f)) of untreated samples (baseline level) from relative Ct-values of treated samples at the indicated time points. RNU2-1f yielded stable concentration upon prolonged storage of CSF at room temperature. The measurements were performed in quadruplicate.

Fig. 2.

Gel electrophoresis of PCR amplification products obtained by qRT-PCR with the Qiagen miR-1246 assay using cDNA from cerebrospinal fluid (CSF) of a patient with primary central nervous system lymphoma (PCNSL) and from CSF of a control patient. Serum was used as template for the positive control reaction. H₂O served as negative control. In CSF, the product of 80 bp corresponding to RNU2-1 fragments was found. The identity of the PCR product in serum was previously confirmed by sequencing.

Fig. 3.

(A) Scatter plots of expression levels of RNU2-1f in cerebrospinal fluid (CSF) samples from patients with primary central nervous system lymphoma (PCNSL) (n = 72) compared with control patients (n = 47), including subgroups of patients with miscellaneous (n = 12) and with inflammatory (n = 35) CNS disorders. Relative expression levels (RELs) of RNU2-1f (y-axis) are normalized to miR-24. The black horizontal lines represent median REL values. Group-wise P values are indicated as determined in Kruskal-Wallis tests with Dunn's multiple comparisons (***P < .001). (B) Receiver-operating characteristic (ROC) curve analyses using relative expression level of RNU2-1f in CSF with an area under the curve (AUC) of 0.909 (95% CI: 0.86–0.96). (C) Scatter plots of expression levels of RNU2-1f + miR-21 in CSF samples from patients with PCNSL (n = 72), compared with control patients (n = 47). Relative expression levels (REL) of RNU2-1f + miR-21 (y-axis) are normalized to miR-24. The black horizontal lines represent median REL values. (B) ROC curve analyses using relative expression level of RNU2-1f + miR-21 in CSF with an AUC of 0.987 (95% CI: 0.973–1.000).

Fig. 4.

CSF RNU2-1f expression levels in time courses during primary central nervous system lymphoma (PCNSL) disease of 9 patients. (A) Five patients (I–V) with complete remission, and (B) 3 patients (VI–VIII) with recurrence after initial remission, and one patient (IX) relapsing after complete remission. The time intervals between the samplings are given in Supplementary material, Table S3. *The first sample of patient IX was collected during initial lymphoma remission.