Genetics of Disease Progression in Diffuse Large B-Cell Lymphoma: Clonal Selection and Acquisition of Newly Acquired Somatic Mutations at Relapse
Authors: Talaulikar Dipti , Jeelall Yogesh , Ziolkowski Andrew , Cook Matthew , Jain Sanjiv , Andrews Dan , Field Matthew , Goodnow Chris 
Published: October 2019

Abstract
Introduction:
Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with ~30% of cases failing treatment or relapsing after R-CHOP chemotherapy. Treatment options for refractory/ relapsed DLBCL are limited and often have a poor outcome. Tumour evolution and selection of a chemo resistant clone are likely explanations making it important to understand clonal heterogeneity at diagnosis and relapse. Green et al have noted a hierarchical acquisition of mutations with disease progression in follicular lymphoma (FL), with BCL2 and CREBBP mutations seen in earlier precursors and MLL2 and TNFRSF14 mutations proposed to be later events. Notably, 1 case analysed at diagnosis and at relapse had variable IgVH indicating origin in a more immature precursors. [Green, M.R., Blood, 2013]. More recent studies indicate that the dominant clone of FL and transformed FL arise by either convergent or divergent evolution from a common mutated precursor through the acquisition of additional genetic events [Pasqualucci, L., Cell Rep, 2014]. Defining the deregulated pathways and oncogenic mutations at diagnosis and later at relapse could help identify novel therapeutic targets for treatment at relapse. We aimed to map clonal evolution at relapse using immunophenotyping, sequencing for the Immunoglobulin variable heavy chain gene (IGHV) and genotyping for known somatic mutations in key lymphoma genes, within matched diagnostic and relapsed DLBCL pairs.
Methods:
Six matched diagnostic and relapsed DLBCL cryopreserved samples were subjected to comprehensive immunophenotyping using 10-colour B-cell flow cytometry panels based on normal ontogeny. PCR on initial and relapsed samples was performed to amplify clonally rearranged IgHV genes from fresh/paraffin embedded primary diagnostic samples. Sanger sequencing was done for IgHV mutation status by targeting the conserved framework regions (FR) FR1 [IGHA: VHFR1-JH] or FR3 [IGHC: VHFR3-JH] using the Invivoscribe kit based on the Biomed-2 protocols. A custom Haloplex library preparation kit designed and ordered from Agilent was used to capture the exons of ~40 genes commonly mutated in DLBCL. Targeted fragments were PCR amplified and target enriched samples sequenced on an Illumina MiSeq sequencer
Results:
Table 1. Immunophenotypic diversity was noted among cases but the phenotype did not change at relapse. IGHV sequencing showed that 3 cases with two distinct lymphoma clones at diagnosis evolved to a single detectable IGHV clonal population at relapse. Lymphoma-associated somatic mutations detected at diagnosis were preserved at relapse and in 4 cases, acquisition of additional mutations was observed.
Table 1: Immunophenotype, IgHV sequence and somatic lymphoma mutations in paired DLBCL samples at diagnosis and relapse Patient number Disease course Immunophenotype IgHV sequence Lymphoma mutations DL157 Diagnosis IgM+, IgD+++, IgG-, CD27-, CD21+++, CD38++, CD10- N/A (not available) EP300 I->V EZH2 D->H NOTCH2 L->H TRAF2 A->T DL157 Relapse Same as diagnosis IgHV3-64 IgHV3-30 EZH2 D->H FOXO1G->D NOTCH2L->HTRAF2 A->T DL214 Diagnosis IgM+++,IgD++, IgG-, CD27-, CD21-, CD38+++, CD10+ IgHV4-30 IgHV4-39 N/A DL214 Relapse Same as diagnosis IgHV4-39 EP300 I->V MYD88 L->P NOTCH1 R->W PRDM1 C->Y SPEN D->E TP53 Y->N DL215 Diagnosis IgM-,IgD- IgG++, CD27++,CD21++, CD38-, CD10- IGHV3-23 IGHV3-23D KMT2D R->C MYD88 L->P NOTCH1 E->K DL215 Relapse Same as diagnosis IGHV3-23 BTG1 H->Y BTG1 Q->Stop BTG2 S->N FOXO1 V->E GNA13 R->H KMT2D R->C MYD88 L->P NOTCH1 E->K PIM1 L->F PIM1 K->N PIM1 V->L DL245 Diagnosis N/A IGHV3-11 IGHV3-33 EP300,I->V,997 NOTCH2,L->V,1559 DL245 Relapse N/A IGHV3-11 EP300,I->V,997 NOTCH2,L->V,1559 DL213 Diagnosis IgM+,IgD-, IgG-, CD27-,CD21+++, CD38+++, CD10++ IGHV3-15 ETS1 A->T EZH2 D->H PRDM1 L->F TNFRSF14 C->S DL213 Relapse Same as diagnosis IGHV3-15 ETS1 A->T EZH2 D->H KMT2D R->Stop PRDM1 L->FSTAT3 R->W DL252 Diagnosis IgM-,IgD++, IgG-, CD27-,CD21-, CD38+, CD10- IGHV1-2 NOTCH2,P->A,2359 TNFRSF14,V->I,241 DL252 Relapse Same as diagnosis IGHV1-2 N/A
Conclusions:
While multiple IGHV sub-clones are present at diagnosis of DLBCL, evolution of a single clone is noted at relapse (convergent model) with acquisition of additional somatic mutations. Immunophenotyping using B-cell maturation flow cytometry panels demonstrates heterogeneity of differentiation between cases but no change in immunophenotype at relapse.

Disclosures
No relevant conflicts of interest to declare.