Integrating genetic subtypes with PET scan monitoring to predict outcome in diffuse large B-cell lymphoma

Abstract

Next Generation Sequencing-based subtyping and interim- and end of treatment positron emission tomography (i/eot-PET) monitoring have high potential for upfront and on-treatment risk assessment of diffuse large B-cell lymphoma patients. We performed Dana Farber Cancer Institute (DFCI) and LymphGen genetic subtyping for the HOVON84 (n = 208, EudraCT-2006-005174-42) and PETAL (n = 204, EudraCT-2006-001641-33) trials retrospectively combined with DFCI genetic data (n = 304). For all R-CHOP treated patients (n = 592), C5/MCD- and C2/A53-subtypes show significantly worse outcome independent of the international prognostic index. For all subtypes, adverse prognostic value of i/eot-PET-positive status is confirmed. Consistent with frequent primary refractory disease, only 67% C2 patients become eot-PET-negative versus 81-88% for other subtypes. Indicative of high relapse rates, outcome of C5 i/eot-PET-negative patients remains significantly worse in HOVON-84, which trend validates in the PETAL and SAKK38-07 trials (NCT00544219). These results show the added value of integrated genetic subtyping and PET monitoring for prognostic stratification and subtype-specific trial design.

Fig. 1. Consort diagram for patient selection and data generation.

^acases of follicular lymphoma grade 3B, primary testicular large B-cell lymphoma, primary CNS large B-cell lymphoma and primary mediastinal B-cell lymphoma were excluded. ^bcases for which FFPE blocks were not available, containing insufficient remaining tissue or not representative material were excluded. ^call cases failing NGS quality control, including tumor percentage <20% as measured by the algorithm ACE were excluded. ^dall cases with stage 1 disease and receiving any other treatments than R-CHOP-14, including Burkitt-like treatment were excluded. NGS=next generation sequencing, i/eot-PET=interim/end-of-treatment ¹⁸fluoro-deoxyglucose-postron emission tomography, IPI=International Prognostic Index.

Fig. 2. Recapitulation of genetic DLBCL subtypes using 716 samples from 3 cohorts by clustering using Non-negative Matrix Factorization (NMF).

Coloured boxes in the heat map and “NMF cluster” bars in the header show the 5 clusters; C1, purple; C2, blue; C3, orange; C4, green; C5, red; and non-annotated samples C0, grey. The header furthermore shows the dendrogram; Top bar “Cohort” colour coded per sample; DFCI, dark red; HOVON-84, blue; PETAL, yellow; “COO” bar, cell-of-origin assignment with GCB, orange; non-GCB, green; unclassifiable, yellow; not assessed, white; “DFCI assignment” bar, DFCI samples with their originally published cluster assignment. “LymphGen” assignment bar, colour coded per sample with colour codes according to their DFCI counterparts with BN2, purple; A53, blue; EZB, orange; ST2, green; MCD, red; Other, grey; unclassified, white. “NMF cluster” bar colour coded accordingly. The right y-axis gives the genetic alterations that define the 5 clusters, per cluster ranked by significance, -log₁₀q Fisher exact test values (Benjamini-Hochberg corrected p-values) grey horizontal bars. Type of genomic drivers are colour coded in the heat map: synonymous mutations, light green; non-synonymous mutations, dark green; copy number gains, red; copy number losses, blue; translocations, purple. Source data are provided in Supplementary Data 1 and 5.

Fig. 3. Association of DLBCL genetic subtypes with PFS and OS.

Kaplan-Meier survival analyses for HOVON-84, PETAL and DFCI cohorts combined, N = 607; (A) PFS for Cell-of origin (COO) class. GCB: green; ABC/non-GCB orange. (B) for OS (C) PFS for DFCI clusters. C1, purple; C2, blue; C3, orange; C4, green; C5, red. (D) for OS (E) PFS for LymphGen classes. P-values were calculated using the log-rank test. BN2, purple; A53, blue; EZB, orange; ST2, green; MCD, red; other, grey. (F) for OS. PFS progression-free survival. OS overall survival. Source data are provided as a Source Data file.

Fig. 4. Bar plots of DFCI subtypes C1-C5 for PET status after 2 cycles of R-CHOP (PETAL), after 4 cycles of R-CHOP (HOVON-84) and at end of treatment (HOVON-84).

C1 patients show early, maximal response after 2 cycles, while only 39%–46% of C3, C4 and C5 patients show early response, which further extends after 4 cycles and until conclusion of treatment. C2 patients show initial similar response, but limited further improvement. PET-negative status: green; PET-positive status red. Source data are provided as a Source Data file.

Fig. 5. Association of DLBCL DFCI-subtype C5 vs. all other with PFS for i/eot-PET negative patients.

Kaplan Meier plots for (A) HOVON-84; (B) PETAL; (C) SAKK-38/07; left column “i-PET2”: i-PET after 2 cycles of R-CHOP, middle column “i-PET4”: i-PET after 4 cycles of R-CHOP, right column “eot-PET”: eot-PET after 6 to 8 cycles of R-CHOP treatment, C5 subtype (red), all other subtypes combined (black), PFS, progression-free survival. The risk table below each plot denotes the number of patients at risk in each group at different timepoints. D Forest plot, per cohort and number of R-CHOP treatment cycles. The groups compared correspond to the groups in (A–C). Hazard ratios, confidence intervals and statistical significance (P-values) were determined using Cox regression. The sample size in each model corresponds to the number of patients at risk at timepoint 0 in the risk tables in (A–C). HR: hazard ratio, black squares with size proportional to weight. (95% CI): confidence interval horizontal lines, and P, unadjusted P-values. Source data are provided as a Source Data file.