Abstract / Summary
Diagnosing rare, aggressive immune-privileged (IP)-large B-cell lymphomas (LBCL), such as primary vitreoretinal lymphoma (VRL) and primary central nervous system lymphoma (CNSL), remains challenging due to the fragility of lymphoma cells within the vitreous or cerebrospinal fluid (CSF) and paucicellular yields, which preclude definitive cytological diagnosis. Clinical MYD88 L265P testing has therefore been incorporated into many haematology diagnostic workflows. These gel-based AS-PCRs are limited by DNA input and quality, are restricted to predefined hotspot variants, and cannot detect CD79B ITAM domain variants, which are seen in IP-LBCLs with MYD88/CD79B (MCD) genotype. To address these limitations, we developed a low-input multiplex MYD88/CD79B PCR and Sanger sequencing workflow that detects canonical MYD88 mutations and additional TIR-domain variants, along with CD79B ITAM-domain mutations, and evaluated its clinical performance in 42 vitreous specimens and 4 CSF specimens. Paired validation with clinical VRL and known inflammatory biopsies ( N = 46) showed higher sensitivity, specificity, and diagnostic accuracy of 81.8%,100%, and 94.7%, respectively, over the current standard of care MYD88 L265P laboratory-developed test (LDT) whilst reducing the minimum DNA input from 1ng to 0.016 ng and maintaining diagnostic concordance with LDT, clinical findings, and whole-exome sequencing (WES). Mixed wild-type and mutant Sanger sequencing peak patterns also provided insight into mutational burden and tumour heterogeneity. Although WES remains more comprehensive, multiplex MYD88/CD79B PCR with sequencing represents a practical intermediate molecular strategy for molecular characterization, identification of the MYD88/CD79B -associated (MCD) genotype, and support for therapeutic and prognostic assessment.