A Reassessment of the Validity of the Routine Lymph2Cx Assay for the Cell-of-Origin Assignment of Diffuse Large...

The Lymph2Cx assay has played a key role in the clinical translation of the diffuse large B-cell lymphoma (DLBCL) molecular classification for cell-of-origin (COO) determination, as mandated by the 2017 Revised WHO Classification. Applicable to routine formalin-fixed paraffin-embedded biopsies, Lymph2Cx has shown high concordance with gold-standard gene expression profiling (GS) performed on fresh/frozen samples. However, conventional concordance metrics may overlook systematic bias. We first reassessed agreement between Lymph2Cx and GS using Bland-Altman analysis and Cohen's kappa. Subsequently, the prognostic impact of the cell-of-origin of diffuse large B-cell lymphoma as determined by Lymph2Cx was compared with that as determined by GS. This was done by assembling large cohorts of publicly available, clinically annotated cases, analyzed by Lymph2Cx or GS, comprising a total of 2321 cases. Lymph2Cx consistently underestimated the frequency of the activated B-cell-like (ABC) subtype, misclassifying a subset of ABC cases as germinal center B-cell-like (GCB) or unclassified (UNC). While ABC DLBCL retained its adverse prognosis across cohorts, GCB and UNC groups defined by Lymph2Cx showed higher hazard ratios than their GS-defined counterparts, consistent with redistribution of higher-risk ABC cases. Overall, although Lymph2Cx remains a valuable tool for routine COO determination, our findings indicate the need for further refinement to improve categorical accuracy, particularly for the ABC subtype, to support optimal clinical risk stratification.