A rapid genotyping panel for detection of primary central nervous system lymphoma

Abstract

Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N = 1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0 to 617 days. Permanent histopathology confirmed PCNSL in 142 of 152 biopsies (93.4%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6% sensitivity and 100% specificity (95% confidence interval [CI]: 44.1% to 70.4% and 87.2% to 100%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8% sensitivity and 100% specificity (95% CI: 56.2%-74.5% and 83.9%-100%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL.

Graphical abstract

Figure 1.

The TetRS qPCR-based rapid genotyping panel detects recurrent molecular alterations relevant to the diverse radiologic differential diagnosis of PCNSL and subsequent diagnostic workup. (A) Clinical characteristics of 159 patients who established a new diagnosis of PCNSL at our facility. Diagnostic assays include all procedures performed at our facility and outside facilities. Time to diagnosis is displayed on a logarithmic scale, calculated from date of admission to our facility. One patient is not displayed, who had 4 lumbar punctures as an outpatient, with the final assay returning positive on the first day of inpatient admission. This cohort included 2 HIV-positive (1.3%), 17 EBV-positive (10.7%), and 13 immunosuppressed patients (8.2%) (supplemental Table 1). (B) Final diagnosis among patients with a new brain lesion with PCNSL in the differential diagnosis. (C) A representative patient with a new brain lesion underwent 2 nondiagnostic lumbar punctures prior to a brain biopsy. Intraoperative histopathology was inconclusive. Chemotherapy was initiated empirically prior to final pathologic diagnosis due to symptomatic decline. Analysis of archived biopsy tissue revealed the MYD88 L265P mutation. (D) Results of TetRS assay on DNA extracts from 71 archived, clinically annotated biopsy specimens by sensitive detection of hotspot mutations in MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF. Expanded results are shown in supplemental Figure 7. EBV, Epstein-Barr virus; Hem., hematologic; HIV, human immunodeficiency virus.

Figure 2.

Detection of PCNSL- and glioma-specific somatic variants with the TetRS rapid genotyping assay in prospectively collected liquid biopsies. (A) The top row of segmented color bars displays the CNS disease status. The next 4 rows represent the results of routine clinical CSF studies and intraoperative frozen histopathology. The bottom row shows the mutant alleles detected in CSF by the TetRS rapid genotyping assay. Expanded results are shown in supplemental Figure 8. (B) Case P22 shows detection of the MYD88 L265P mutation in CSF from a patient with a cerebellar mass, who underwent gross total resection due to nondiagnostic intraoperative frozen specimen analysis. Cytology and permanent histologic analysis revealed PCNSL 4 days postoperatively. (C) Case P7 shows detection of the TERT C228T mutation in the CSF of a patient with a right temporal mass. Surgical needle biopsy was concluded after intraoperative frozen analysis showed high-grade glioma but converted to open craniotomy and subtotal resection due to intraoperative bleeding. GBM, glioblastoma multiforme; HGG, high-grade glioma.