Genotyping on ctDNA Identifies Shifts in Mutation Spectrum Between Newly Diagnosed and Relapse/Refractory DLBCL

Abstract

Purpose: Diffuse large B cell lymphoma (DLBCL) is an aggressive B-cell malignancy with clinical and molecular heterogeneity whose genetics may have clinical implications for patient stratification and treatment. The circulating tumor DNA (ctDNA) is a novel noninvasive, real-time, and tumor-specific biomarker harboring tumor-derived genetic alterations that are identical to those of tumor cells, thus showing great promise in individualized medicine, including precise diagnosis, prediction of prognosis, response monitoring, and relapse detection for DLBCL.

Patients and methods: In this study, we applied NGS analysis to tumor biopsies and ctDNA samples from 16 DLBCL subjects. Then, we compared the genomic alterations from 41 newly diagnosed patients and 56 relapsed/refractory (R/R) patients.

Results: Our results show that ctDNA can function as a liquid biopsy for tracking recurrently mutated genes in DLBCL (sensitivity: 87.50%). The mutational profiles of newly diagnosed and R/R DLBCL groups largely overlapped, but the frequencies of some gene mutations differ between the two cohorts. The distribution of mutations also revealed different frequencies in the two cohorts due to different signaling pathways. Genes from apoptosis pathway, immune response and BCR pathway suffered more mutations in R/R patients.

Conclusion: Overall, this study establishes ctDNA as an easily accessible source of tumor DNA for DLBCL genotyping and provides a deeper understanding of the somatic alteration spectrum for both newly diagnosed and R/R DLBCL patients.

Keywords: circulating tumor DNA; diffuse large B cell lymphoma; liquid biopsy; mutation; next-generation sequencing.

Figure 1

ctDNA genotyping discloses somatic mutations in DLBCL. (A). Number of mutations in a given patient detected in plasma ctDNA and/or tumor gDNA. (B). Venn diagram summarizing the overall number of mutations discovered in both plasma ctDNA and tumor gDNA. (C). The corresponding overall sensitivity of plasma ctDNA genotyping in discovering biopsy-confirmed mutations.

Figure 2

Percentage of biopsy-confirmed mutations identified in ctDNA according to mutated allele frequency in biopsy. (A). The mutation abundance in plasma cfDNA vs the mutation abundance in tumor gDNA is comparatively represented in the scatter plot for each variant identified in the discovery cohort. (B). ROC analysis illustrates the performance of plasma cfDNA genotyping in detecting biopsy-confirmed tumor variants according to the variant allele frequency of mutations in tumor gDNA in the discovery cohort. The bar graph shows the allele frequency in tumor gDNA of the variants that were discovered in plasma cfDNA (black bars) or missed in plasma cfDNA (gray bars). The dash line tracks the 30% variant allele frequency threshold.

Figure 3

Number and type of nonsynonymous somatic mutations identified in each gene.

Figure 4

Mutation distribution among DLBCL patients. (A). Distribution of mutations identified in primary and relapse/refractory DLBCL samples. (B). Distribution of mutations identified in DLBCL samples according to COO classification.

Figure 5

Distribution of mutations according to signaling pathways in primary and relapse/refractory DLBCL samples.

Figure 6

Distribution of mutations according to clinical characteristics. (A). Distribution of mutations according to IPI. (B). Distribution of mutations according to complications. (C). Distribution of mutations according to Ann Arbor stage. (D). Distribution of mutations according to extranodal involvement.