Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens

Abstract

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.

Graphical abstract

Figure 1.

Schematic overview of the study design. For details on clinical characteristics and pathological features of the cases refer to supplemental Tables 1 and 2. QC, quality control.

Figure 2.

Performance of the Lymph3Cx assay in the validation cohort and inter-laboratory concordance. (A) Heatmap of gene expression in the independent validation cohort of 88 pathologically defined PMBCL cases and 70 DLBCL cases. Each column represents an individual case, and the discriminating gene features included in the Lymph3Cx assay are ordered in rows. The top 6 genes are overexpressed in DLBCL, and the other 24 show higher expression levels in PMBCL. Housekeeping genes and the features used for DLBCL COO assignment are not displayed for visualization purposes. Cases are ordered according to their respective model score, with cases that obtained the highest score on the right. (B) Displayed are only the pathologically defined PMBCL cases with their respective assignment by the molecular gene expression–based assay (Lymph3Cx). No bias was observed with regards to mediastinal/nonmediastinal location of biopsy site, providing evidence that the assay is also applicable to PMBCL(-like) cases arising outside the mediastinum or nonmediastinal biopsy specimens, respectively. Interestingly, the 4 cases with a molecular signature of DLBCL (box) did not harbor chromosomal rearrangements of CIITA and/or the programmed death ligand (PDL) loci, or PDL copy-number (CN) alterations. (C) Distribution of model scores in the validation cohort. (D) Comparison of Lymph3Cx scores for selected cases of the validation cohort from 2 independent laboratories (BC Cancer Agency [BCCA] and Mayo Clinic). Dotted lines represent the defined thresholds to discriminate PMBCL (dark blue) from DLBCL (brown) using the Lymph3Cx assay (uncertain category displayed in purple). Of note, no case changed subtype assignment between the different laboratories. amp, amplification; ba, break-apart; CNA, copy-number alteration; N/A, not assessable.