Abstract P5-04-13: Triple negative breast cancer: Expression of immuno-oncology treatment targets and prevalence of tumor infiltrating lymphocytes in paired primary tumors and metastases
Authors: Stovgaard Elisabeth S , Poulsen Tim S , Balslev Eva , Nielsen Dorte L , Roslind Anne , Talman Maj-Lis , Kümler Iben , Christensen Ib J , Høgdall Estrid 
Published: March 2020

Introduction: There is extensive research into immune-oncology (IO) treatment targets for triple negative breast cancer (TNBC). However, little knowledge exits on variations in expression levels of IO targets between paired primary tumors (PT) and metastases (MT) in TNBC.

Objectives: To compare expression levels of IO drug targets in paired PT and distant MT from TNBC patients. PD-L1 protein expression was examined immunohistochemically, as well as differences in levels of tumor infiltrating lymphocytes (TILs).

Material and methods:552 patients were primarily operated for TNBC at 2 university hospitals in Denmark between 2004 and 2014. Of these, samples from 22 patients were available from both PT (22 samples) and distant MT (24 samples, with 2 chronologically separate metastases for two patients). Localizations of metastases included 9 brain, 1 skin and 1 lymph node (both not regional), 2 bone, 5 liver, 2 soft tissue, 1 lung, and 1 adrenal gland. Gene expression analyses were performed on the Ion Torrent S5XL system, using the Oncomine Immune Response Research Assay. PD-L1 expression was evaluated immunohistochemically using the 28-8 clone (pharmdX). Cut-off levels for PD-L1 expression were 1% positive tumor cells. TILs were evaluated in accordance with guidelines from the International Immuno-Oncology Biomarker Working Group.

Results: Gene expression levels of 29 established IO drug targets were tested on 18 of the 22 TNBC patients with matched PTs and MTs (Table 1). 4 genes showed significant downregulation in MTs compared to PTs: IDO1 (p=0.01), CXCR4 (p=0.01), KIR2DL1 (p=0.05) and TNFRSF9 (p=0.03). However, other important IO targets such as CTLA4, STAT3, TIGIT, and TNSFRSF18 (GITR) were not significantly different between PT and MT. CD276 (B7-H3), PD-1, IL2 and LAG3 showed a tendency towards significance (p= 0.08, 0.07, 0.05, 0.08), with LAG3 being the only gene upregulated in MT compared to PT. TILs were evaluated for all PTs and MTs. TILs in PTs ranged from 1 to 40% (median=13%). TILs in MTs ranged from 1 to 60% (median=5%). There was no significant overall change in TILs from PT to MT (p=0.2). However, 9 patients had a difference in TILs between PT and MT of &gt; ±10% (-20 - +30).

PD-L1 immunohistochemical evaluation was possible for both PT and MT in 20 cases. There was no significant overall change in PD-L1 expression (p=0.39). However, 4 patients with positive PTs, had negative MTs, and 4 patients with PD-L1 negative PTs, had positive MTs.

Discussion: This is, to our knowledge, one of the largest studies of gene expression of IO drug targets in TNBC matched PTs and MTs. We show significant differences in gene expression levels for IDO1, CXCR4, KIR2DL1 and TNFRSF9, pointing to a generally less immune-active environment in MTs. This was also corroborated by lower levels of TILs in MTs, although these findings were not significant. For PD-L1 protein expression, we found that PD-L1 expression can change between PT and MT. In the clinical setting, treatment decisions are often based upon expression levels in a single biopsy or surgical specimen. Intra-patient heterogeneity should be taken into account in the diagnostic setting, and when making treatment decisions.

Table 1IO drug targetRatio (Primary/metastasis)P-valueIDO19.65 (1.68-54.95)0.01CXCR41.83 (1.17-2.87)0.01KIR2DL12.17 (1.01-4.69)0.05TNFRSF97.11 (1.21-41.93)0.03CD276 (B7H3)1.51 (0.94-2.39)0.08PDCD1 (PD1)3.32 (0.88-12.64)0.07IL22.87 (0.99-8.28)0.05LAG30.28 (0.07-1.16)0.08CTLA41.96 (0.45-8.51)0.35STAT31.21 (0.92-1.60)0.16TIGIT1.87 (0.25-14.03)0.53TNFRSF18 (GITR)0.38 (0.06-2.20)0.26

Citation Format: Elisabeth S Stovgaard, Tim S Poulsen, Eva Balslev, Dorte L Nielsen, Anne Roslind, Maj-Lis Talman, Iben Kümler, Ib J Christensen, Estrid Høgdall. Triple negative breast cancer: Expression of immuno-oncology treatment targets and prevalence of tumor infiltrating lymphocytes in paired primary tumors and metastases [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 P5-04-13.