Abstract P4-05-09: Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes
Authors: Licata Luca , Galbardi Barbara , Győrffy Balázs , Karn Thomas , Sica Lorenzo , Rognone Alessia , Zucchinelli Patrizia , Aldrighetti Daniela , Zambelli Stefania , Gianni Luca , Bianchini Giampaolo 
Published: March 2020

Abstract
Background. High-TMB (HTMB) is an emerging promising agnostic biomarker for predicting benefit from immune-checkpoint inhibitors, independently of tumor type. At ASCO 2019, the TAPUR trial reported an interesting 21% ORR in heavily pretreated metastatic BC patients with very HTMB [vHTMB, ≥9 mutations/megabase (Muts/Mb)]. We aimed to define the differential gene expression and methylation landscape between low and high TMB in each BC subtype. Methods. In TCGA, we identified 848 patients with WES data available for TMB estimation. [ER+/HER2- (LumA by PAM50)) n = 364; ER+/HER2- (LumB by PAM50), n = 147; HER2+, n = 158; and TN, n = 179]. High TMB was defined according to two different cut-offs: ≥9 (vHTMB) and≥5 Muts/Mb (HTMB). The second arbitrary cut-off was used to define a larger group allowing to better characterize the different molecular landscapes associated with high and low TMB in each BC subtype. The HTMB group was compared with an equal number of tumors with low TMB. We assessed the differential RNA expression and methylation of single genes and pathways (defined using Gene Ontology - GO). “Common” genes and pathways were defined as recurrently associated with TMB (p&lt;0.05) in all subtypes and with a combined p value ≤0.00001. Results. The overall prevalence of vHTMB (≥9) was 4.5%, with no substantial differences across subtypes (4.4%, LumA; 4.8% LumB; 5.7% HER2+; 3.3% TN). The prevalence of HTMB (≥5) was 13.7%, but it was different across BC subtypes (p=8.0E-07) (8.2%, LumA; 12.9% LumB; 13.3% HER2+; 25.7% TN). We found more “common” genes down-regulated (n=70) than up-regulated (n=3) in HTMB group. Two of these three genes (HSPE1 and FEZF1.AS1) have been associated with poor prognosis in BC. When we considered the “common” pathways, only 3 were up-regulated in HTMB, all implicated in post-transcriptional repression of gene expression (gene silencing by miRNA and mRNA binding involved in post-transcriptional gene silencing). Conversely, 66 were significantly down-regulated (including transcription coregulator and coactivator activity, protein serine/threonine kinase activity and ubiquitin-protein transferase activity and binding). Some genes and pathways were associated with TMB only in a specific BC subtype (p ≤0.00001). For instance, 16 pathways were down-regulated in the HTMB group of TNBC. These inlcuded 12 pathways implicated in immune response. Conversely, in LumB, 11 pathways were up-regulated in HTMB group and implicated in immune response. Intrestingly, these pathways were all significantly down-regulated in the HTMB group of LumA and TN. In HTMB group, we found 7 and 4 “common” genes hypermetylated and hypomethylated, respectively. Four pathways were commonly hypermethylated (chromatin silencing at rDNA, telomere organization and positive regulation of G1/S transition of mitotic cell cycle) and five were hypomethilated (including mitotic sister chromatid segregation). Considering private alterations, in TNBC, 23 of 27 significant pathways were hypermetylated in HTMB group including double-strand break repair via nonhomologous end joining, epigenetic negative regulation of gene expression, and regulation of gene silencing by miRNA. Conclusions. Very-high TMB which is considered potentially druggable (≥9 Muts/Mb) is rare in BC, and equally frequent in all subtypes. Instead, HTMB (≥5 Muts/Mb) is more frequent in TNBC. BCs with HTMB had a different molecular landscape. Overall, several genes are recurrently down-regulated in HTMB group, and this is at least partly due to miRNA regulated post-trascriptional silencing, which might rapresents a new mechanism of immune escape. The positive association between TMB and immune genes in LumB, as well as the negative association in TN and LumA, suggest that immune editing and surveillance might be dependent on the molecular context.
Citation Format: Luca Licata, Barbara Galbardi, Balázs Győrffy, Thomas Karn, Lorenzo Sica, Alessia Rognone, Patrizia Zucchinelli, Daniela Aldrighetti, Stefania Zambelli, Luca Gianni, Giampaolo Bianchini. Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-09.