CD28 Mutations in Peripheral T-Cell Lymphomagenesis and Progression
Authors: Rohr Joseph , Guo Shuangping , Hu Dongdong , Bouska Alyssa , Gascoyne Randy D. , Rosenwald Andreas , Simone Peter , Zhang Weiwei , Xiao Wenming , Wang Chao , Fu Kai , Greiner Timothy C. , Weisenburger Dennis D. , Vose Julie M. , Staudt Louis M. , Berger Francoise , Borgstahl Gloria , Davis Simon , McKeithan Timothy , Iqbal Javeed , Chan Wing-Chung (John) 
Published: October 2019

Abstract
Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of diseases with poor prognosis and no standard therapy due to widely varying responses to treatments, thus new therapeutic targets need to be identified. Gene expression profiling (GEP) has helped to separate classes of these diseases to predict survival. Recent genetic studies on the two most common subtypes of PTCL in the Western world, angioimmunoblastic T-cell lymphoma (AITL) and PTCL, not otherwise specified (PTCL-NOS), have revealed recurrent mutations in the epigenetic modifiers TET2, IDH2, and DNMT3A; in the small GTPase RHOA; and rarely in the T-cell receptor (TCR) adaptor protein FYN. Because TCR stimulation is necessary for normal T-cell expansion, activating mutations in the stimulation (CD3/TCR) and costimulation (CD28) pathways could be involved in malignant lymphopoiesis.
We performed whole-transcriptome sequencing on a small set of primary PTCL cases and found mutations in CD28, including an aspartate 124 mutation within a fusion transcript between CD28 and family member ICOS as well as a threonine 195 mutation. On targeted re-sequencing of 92 PTCL cases, we found two AITL cases and one ALK- anaplastic large cell lymphoma case with an aspartate 124 mutation (variant frequencies [VF]: 1.03% to 5.90%). We also found mutations at threonine 195 (two AITL, one PTCL-NOS, Tbx21 subtype; VF: 2.90% to 12.30%). Additionally, we found two recurrent mutations with low variant frequencies (<2.0%). We also determined each targeted sequencing sample’s RHOA mutation status; high-VF CD28 mutations also had RHOA glycine 17 mutations, almost all of which were to valine (G17V) consistent with previous reports of RHOA mutations in PTCLs.
Surface Plasmon Resonance (SPR) analysis of the two highest-VF mutated residues D124 and T195 in CD28 show increased affinities of CD28 mutants for partners. The D124 to valine mutation (D124V) within the extracellular ligand binding region increases affinity for CD86. The T195 to proline mutation (T195P) increases binding of GRB2 and GADS/GRAP2 to the CD28 cytoplasmic tail at the intracellular SH2-binding domain. Molecular modeling of these mutations offers possible explanations for these differential affinities, including potential increased electrostatic binding at the extracellular interface and binding partner side-chain stabilization intracellularly. Further, these mutations have a significantly increased capacity to activate NF-κB upon ligation of CD80 or CD86 and anti-CD3 stimulation.
Mutations in CD28 often occur with low frequency in PTCL tumor samples; given this low frequency, the mutations are likely not a primary mechanism of lymphomagenesis. It is possible that over-activation of CD28 represents a late-stage event in clonal evolution, allowing a tumor subclone to further grow and proliferate. These CD28-mutant clones may play a role in later disease course, in refractory disease, or in post-therapeutic relapse.

Disclosures
Fu: Nanostring: The author is a potential inventor on a patent application using Nanostring technology for the Lymph2Cx assay, which has been licensed from the NIH by Nanostring Patents & Royalties.