The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well-known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.

In routine cancer molecular pathology, various independent experiments are required to determine mutation and amplification status of clinically relevant genes. Most of these tests are designed to identify a limited number of genetic aberrations, most likely in a given tumor type. We present a modified version of a multiplexed PCR and IonTorrent-based sequencing approach that can replace a large number of existing assays. The test allows for the simultaneous detection of point mutations and gene amplifications in 40 genes, including known hotspot regions in oncogenes (KRAS, BRAF), inactivating mutations in tumor suppressors (TP53, PTEN), and oncogene amplifications (ERBB2, EGFR). All point mutations were confirmed using certified diagnostic assays, and a sensitivity and specificity of 100% (95% CI, 0.875-1.0) and 99% (95% CI, 0.960-0.999), respectively, were determined for amplifications in FFPE material. Implementation of a single assay to effectively detect mutations and amplifications in clinically relevant genes not only improves the efficiency of the workflow within diagnostic laboratories but also increases the chance of detecting (rare) actionable variants for a given tumor type that are typically missed in routine pathology. The ability to obtain comprehensive and rapid mutational overviews is key for improving the efficiency of cancer patient care through tailoring treatments based on the genetic characteristics of individual tumors.

Sensitivity for detecting mutations was highly comparable among all platforms. The choice for either a dedicated genotyping platform or an NGS platform is basically a trade-off between speed and genetic information.

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