Single cell DNA analysis of MYD88^L265P mutation reveals mutational heterogeneity and is more sensitive and specific than cell-free and cellular DNA analysis for the diagnosis of vitreoretinal and central nervous system lymphoma

Abstract

Purpose : MYD88^L265P mutation occurs in high frequency in vitreoretinal (VRL) and central nervous system lymphoma (CNSL), up to 70-75%. Single cell (sc), cell-free (cf) and cellular (bulk) DNA analyses can be used for detecting MYD88 ^L265P mutations. This study compares diagnostic accuracy of these methods in MYD88 ^L265P mutation detection.

Methods : This study was performed with informed consent and institutional review board approval. A total of 3 vitreous and 3 CSF were obtained from 5 patients with clinically proven VRL and CNSL respectively (Table 1). Patient 1 had a post treatment sample of vitreous after clinical resolution to serve as a true negative (TN) sample. Single cell (sc), cell free (cf) and bulk cellular DNA analysis of MYD88^L265P was performed using published methods. We previously established a cut-off value >9.5% homozygous MYD88^L265P to define the presence of a MYD88^L265P mutation. Measures of diagnostic accuracy included sensitivity and specificity.

Results : Sc MYD88 analysis correlated with clinical stage of disease activity and resolution, (5 true positive, TP samples and 1 TN sample) with 100% sensitivity and specificity. In contrast, cf- and cellular DNA analysis both showed only 40% sensitivity but 100% specificity (1TP, 1 TN and 3 false negative, FN) when compared to disease status. Sc-MYD88 DNA analysis revealed MYD88^L265P mutation detection failed in both cf- and cellular DNA analysis when the homozygous percentages were low (0 to 14%). MYD88 ^L265P mutation was only detected in cf- and cellular DNA analysis when the homozygous mutation population was ≥75%.

Conclusions : Although cf- and cellular DNA analysis of MYD88 ^L265P remains a useful adjunctive test for VRL/ CNSL diagnosis that is accessible for most labs, reduced detection sensitivity may occur when the percentage of homozygous mutation is low. Sc-MYD88 ^L265P DNA analysis, although technically more challenging is superior in sensitivity and specificity as it also reveals the MYD88 ^L265P mutation heterogeneity. Furthermore, changes in sc-MYD88 ^L265P mutational zygosity with treatment can also be monitored with the single cell DNA approach.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Table1: Comparison of single cell, cell free and cellular (bulk cell) MYD88L265P DNA analysis with disease status.