Targeted sequencing in DLBCL, molecular subtypes, and outcomes: a Haematological Malignancy Research Network report

Abstract

Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.

Graphical abstract

Figure 1.

Heat map of characteristic mutations from each of the 6 genetic clusters that were identified by using the Akaike information criterion in the analysis cohort (n = 928). Distinct clusters are identified by color. Along the bottom of the figure, the color strip shows the corresponding cell-of-origin classification for each patient. The panel on the right-hand side shows the enrichment for mutations within each cluster, with a logarithmic q-value scale. Only those mutations are shown that are identified as significantly enriched for the given group, as determined by a Benjamini-Hochberg adjusted q < 0.05 from a χ² test of independence. “HD” indicates the homozygous deletion or a mutation in this gene; “noncan” denotes a non-canonical mutation; and “amp” indicates an amplification. For cell-of-origin, UNC represents the unclassified group.

Figure 2.

Relations between gene expression profiling and the 6 genetic clusters that were identified by using the Akaike information criterion for the 519 subjects with available gene expression profiling data. (A) Heat map of a selection of signatures. (B) Highlights of a small number of these that exhibit the strongest trends.

Figure 3.

Survival in clusters identified by using the Akaike information criterion. (A) OS of the 690 patients treated with curative intent stratified according to cluster. (B) OS of the 579 de novo DLBCL NOS patients treated using R-CHOP, stratified according to cluster. (C) Progression-free survival of the 690 patients treated with curative intent stratified according to cluster. (D) Progression-free survival of the 579 de novo DLBCL NOS patients treated by using R-CHOP, stratified according to cluster. (E) Crude hazard ratios of cluster membership for the 579 de novo DLBCL NOS R-CHOP–treated patients. (F) Adjusted hazard ratios of the subset of the group of 579 patients with IPI data available (n = 499). CI, confidence interval.

Figure 4.

OS of the de novo DLBCL NOS R-CHOP–treated patients (n = 579), stratified according to a selection of genetic features. (A) NOTCH1 mutation. (B) MYC rearrangement status in the subset of patients belonging to the BCL2 Akaike information criterion subtype. (C) TP53 mutation or homozygous deletion. (D) The effect of TP53 mutation or homozygous deletion in each genetic subtype.