Defining Immunoglobulin Somatic Hypermutation in De Novo Diffuse Large B-Cell Lymphoma Patients: Potential Application for Prognosis and Risk Stratification

Context

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of diseases with variable clinical presentation, morphologic features, genomics, gene expression signature and prognosis. Some of the variability in patient course and response to therapy is likely to represent a function of developmental stage and/or specific pathway of transformation. We have been engaged in a detailed investigation of the molecular and clinical features of a large cohort of patients with DLBCL at the MD Anderson Cancer Center. Through these analyses we have begun to subcategorize this patient population based on these distinctive clinical and biological parameters. In this current aspect of our investigation we have explored the prevalence of somatic hypermutation (SHM) of the immunoglobulin loci in these de novo DLBCL patients using the platform of multiplex PCR and high-throughput sequencing (immunoSEQ) developed by Adaptive Biotechnologies, Inc. It has previously been established that the presence or absence of somatic hypermutation is an independent prognostic factor in patients with chronic lymphocytic leukemia (CLL). The ultimate goal of this collaborative effort is to determine if a similar biological mechanism between somatic hypermutation and prognosis exists within the population of DLBCL patients or subset and to relate the presence of SHM to clinical, pathological, and molecular aspects of this disease.

Objective

In this study, we investigated whether the immunoSEQ (Adaptive Biotech) assay could be used to reliably discriminate dominant clones in diagnostic specimens from patients with DLBCL with regard to rearrangement status of the immunoglobulin heavy and light (kappa and lambda) chains and the presence or absence of SHM.

Patients

The study group consisted of 200 DLBCL patients treated with R-CHOP. Patients with primary mediastinal large B-cell lymphoma, primary cutaneous DLBCL, primary central nervous system DLBCL, and DLBCLs transformed from a low-grade B-cell lymphoma or associated with HIV infection were excluded.

Methods

Genomic DNA was extracted from FFPE sections of diagnostic lymph node specimens of patients with DLBCL. Immunoglobulin heavy and light chain sequences were then independently amplified using multiplex PCR with optimized primer sets. Following high-throughput sequencing, a bioinformatics pipeline clusters the sequences into distinct clonotypes to determine overall frequencies and to identify diagnostic clones. V, (D,) and J genes are also identified for each clonotype, and point mutations that are not known germ-line allele variants are assigned as somatic hypermutation events.

Results

Using both the IgH and IgL (kappa and lambda) we have been able to identify an index trackable sequence in 90%+ of the samples (we identify an index diagnostic sequence or sequences in about 70% of the cases using each assay individually). Using a definition of SHM as >2% point mutations in the observed V gene, the samples can be split into three distinct categories: 1, V(D)J or VJ rearranged with SHM (50-55%); 2, V(D)J or VJ rearranged without SHM (10-25%) and 3, DJ only evident (20-40%)

The vast majority of complete V(D)J rearrangements are in-frame.

Conclusions

The IgH and IgL immunoSEQ assays are robust in their ability both to identify dominant sequences in diagnostic lymph node specimens from patients with DLBCL and to distinguish those clones in which evidence of somatic hypermutation is present. The distribution of SHM in these samples lends itself to potential correlative and stratifying analyses on this well-characterized patient cohort, and likely have significant application in other aggressive B-cell lymphoma patients.