Unraveling CLL progression in unmutated patients: linking functional AID expression with disease evolution

OPPEZZO PABLO; SEIJA, NOÉ; PRIETO DANIEL; JULIETA SPULVEDA; PABLO MORANDE; URIEPERO, ANGIMAR; NAVARRETE MARCELO

Nueva York

Workshop; XVII International Workshop on Chronic Lymphocytic Leukaemia; 2017

iwCLL Consortium

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes,which are critically important for an effective immune response development. In addition, AID seems to contribute to B cell tolerance in mice and humans eliminating developing autoreactive B cells. As a trade-off of the beneficial roles of AID physiological activity, this enzyme can also contribute to cellular transformation and tumor progression through its off-target mutagenic activity. Mounting evidences indicate a role for AID in disease progression and poor prognosis of chronic lymphoid neoplasms. Since these leukemias are not immediately derived from germinal center B cells, normal AID regulation might not be fully functional. Specifically, in CLL we have shown that AID isanomalously expressed in the peripheral blood of patients with Unmutated (Um) VH genes, correlating with active CSR and poor clinical outcome (Oppezzo et al., Blood 2005). In addition, others and us have described that AID expression is mainly restricted to distinct proliferative subsets within the leukemic clone(Albesiano et al., Blood, 2003; Palacios et al., Blood, 2010 and Patten et al., Blood, 2012). Despite the fact that AID expression has been associated with adverse cytogenetics and worse outcome (Leuenberger, M., XVII INTERNATIONAL WORKSHOP ON CHRONIC LYMPHOCYTIC LEUKEMIA 2017 207 Downloaded by [University of Florida] at 09:55 17 December 2017 Mod Pathol., 2010) definitive experimental demonstration that AID contributes to CLL progression is missing. To obtain insight into this issue we analyzed mutation load and signatures in Um patients in which anomalous expression of AID in peripheral blood was sustainedthroughout disease evolution. We compared the mutational profile by Whole Exome Sequencing(WES) of purified CD19þ cells extracted from 5 progressivepatients expressing AID (Um/AIDpos) andfrom 5 progressive patients in which AID was notdetected (Um/AIDneg). Samples were obtained atdiagnosis and at second time point after the first treatment(AFT). Analysis was performed according toGATK-best practice guidelines, variant discovery wasperformed by Varscan algorithm. Unsupervised AIDmutational profile was assessed by the Bayesian nonnegativematrix factorization algorithm as previouslydescribed (Alexandrov et. al., Nature, 2013), with thisstrategy we identified non-canonical AID signatures(nc-AID), i.e. A>C mutations at WA motifs in sensestrand and T>G mutation at TW motifs in antisensestrand. For supervised analysis of canonical AID signatures(c-AID) we quantified C to T mutations at WRCYmotifs in the sense strand or T to C variants in YGRWmotifs in antisense strand.We found that the total number of somatic mutationsis significantly increased in Um/AIDpos compared withUm/AIDneg patients independently of the diseasetime of the analyzed sample. A higher frequency ofsomatic mutations were observed in Um/AIDpos samplesat debut of the disease compared with Um/AIDpos samples AFT. We next analyzed mutation signaturesin the different groups: Um/AIDpos at diagnosis;Um/AIDpos AFT; Um/AIDneg at diagnosis and Um/AIDneg AFT. Our results show no significant differencesobserved between the groups concerning c-AIDmutations. However, an increased and significant differencewas found in the group Um/AIDpos comparedwith Um/AIDneg. Interestingly, a higher frequency ofnc-AID mutations was observed in Um/AIDpos groupat diagnosis time compared with Um/AIDpos AFT,which might suggest selection for samples with lowerAID levels and/or mutation loads. In addition, theunsupervised analysis identified the presence otherpreviously described signatures (Alexandrov et. al.,Nature, 2013), such as Aging, and APOBEC in all samples.We also analyzed AID-catalyzed mutations thatwere found at the debut of the disease and maintainedduring disease evolution, as well as those thatappeared only in the AFT samples. Presently, mutationsin selected genes are being confirmed in a largercohort. We will discuss these results.AID expression could be specially problematic inchronic diseases, where even a small but continuouslevel of AID activity can lead to selectable geneticmutations over time, giving rise to more aggressivetumors and treatment resistance. Our results supportthis notion and describes for the first time the mutationalprofile of Um and progressive patients withanomalous AID expression through their evolution.Under the light of the hypothesis that AID expressionis a hallmark of an activated microenvironment resultingin disease progression our results will allow us toidentify AID mutated genes which could be implicatedin CLL tumor progression.This work was supported by ANII. ProyectoFMV_1_2014_1_104397 and CSIC-Udelar. Programa deIþD 2014.