Abstract S1-08: Reduced tumor lymphocytic infiltration in the residual disease (RD) of post-neoadjuvant chemotherapy (NAC) triple-negative breast cancers (TNBC) is associated with Ras/MAPK activation and poorer survival
Authors: Balko Justin M , Denkert Carsten , Salgado Roberto , O'Hely Martin , Savas Peter , Beavis Paul A , Darcy Phil K , Combs Susan , Rimm David L , Giltnane Jennifer M , Estrada Monica V , Sanders Melinda E , Cook Rebecca S , Wang Kai , Miller Vincent A , Stephens Phillip J , Yelensky Roman , Pinto Joseph A , Doimi Franco , Gomez Henry , Arteaga Carlos L , Loi Sherene 
Published: May 2015

Abstract
Backgound: Tumor-infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBCs, with several retrospective analyses demonstrating that TNBCs with high baseline TILs have higher rates of pathologic complete response (pCR) to NAC. Moreover, the TIL burden in the RD of patients who do not achieve pCR to NAC is also correlated with prognosis. However, insight into the molecular pathways in TNBC which modulate heterogeneity in host anti-tumor immune responses is lacking. To address this gap in knowledge, we analyzed TILs retrospectively in a cohort of clinically and molecularly characterized TNBCs with RD after NAC.
Methods: TILs were scored in H&E stained slides by expert pathologists in the post-treatment tumors of 92 NAC-treated TNBC patients with RD at the time of resection and in 44 matched baseline diagnostic biopsies. Genomic alterations in the RD were assayed using targeted next-generation sequencing (tNGS) while selected transcriptional signatures were evaluated by NanoString as previously published (Balko et al, Cancer Discovery 2014). Differences in pre- and post-NAC TILs were compared between tumors harboring alterations in cell cycle, PI3K/mTOR, growth factor receptors, Ras/MAPK and DNA repair pathways. Associations of TILs with transcriptional signatures were also tested.
Results: A strong positive association of TILs in NAC-treated specimens was observed with RFS (coxPH p=0.0001, relative risk reduction of 3.4% for each % of TILs) and OS (p=0.0016; relative risk reduction of 2.8% for each % of TILs). In multivariate analysis with stage, age, node status and RD tumor cellularity, TILs in the post-NAC disease remained a significant predictor of RFS and OS (p=0.0008 and p=0.007, respectively). TILs tended to decrease with NAC in paired samples, although this decrease was not statistically significant (p=0.07).
Genetic alterations in the Ras/MAPK (amplifications in KRAS, BRAF, RAF1 and truncations in NF1) and cell cycle pathway (alterations in CCND1-3, CDK4, CDK6, CCNE1, RB, AURKA and CDKN2A) were associated with lower TILs in RD (p=0.005 and p=0.05, respectively). A significant inverse linear correlation was detected between a transcriptional signature of Ras/MAPK activation (Pratilas et al, PNAS 2009) and TILs in the RD (Spearman’s r=-0.42; p=0.00028). Total number of alterations of likely functional significance detected by tNGS showed no association with TILs, suggesting that the association of Ras/MAPK deregulation and cell cycle alterations with TILs may be a pathway-specific effect.
In TNBC cell lines, chemical inhibition of MEK transcriptionally up-regulated MHC-I and MHC-II molecules, while simultaneously down-regulating mRNA expression of the immune checkpoint inhibitor PD-L1 (MDA-231 p=0.00002, BT549 p=0.0003, and SUM159PT p=0.009). In vivo experiments confirming these associations are underway.
Conclusions: Our data suggest a strong correlation of Ras/MAPK pathway activation with immune-evasion and outcome in TNBC. With additional mechanistic understanding, rational design of clinical trials combining MEK inhibitors with PD-L1 antibodies in TNBC may be warranted.
Citation Format: Justin M Balko, Carsten Denkert, Roberto Salgado, Martin O'Hely, Peter Savas, Paul A Beavis, Phil K Darcy, Susan Combs, David L Rimm, Jennifer M Giltnane, Monica V Estrada, Melinda E Sanders, Rebecca S Cook, Kai Wang, Vincent A Miller, Phillip J Stephens, Roman Yelensky, Joseph A Pinto, Franco Doimi, Henry Gomez, Carlos L Arteaga, Sherene Loi. Reduced tumor lymphocytic infiltration in the residual disease (RD) of post-neoadjuvant chemotherapy (NAC) triple-negative breast cancers (TNBC) is associated with Ras/MAPK activation and poorer survival [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-08.