Abstract P4-04-07: Association between tumor infiltrating immune cells and disseminated tumor cells in the bone marrow of patients with primary breast cancer: A ten year follow-up
Authors: Kasimir-Bauer S , Spiekermann A , Friedrich CM , Hoffmann O , Schmid KW , Kimmig R , Bankfalvi A 
Published: May 2016

Abstract
Background: Disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients (pts) were shown to be an independent prognostic factor with regard to reduced PFS and OS. Although this negative prognostic impact has been related to chemotherapy resistant and stem cell-like cells, the microenvironment of the primary tumor might also influence tumor cell spread and thus, outcome of the disease. After a ten year follow-up, we here analyzed the prognostic significance of infiltrating leukocytes in the tumor (TILs) and stroma (SILs) with regard to PFS and OS as well as their association with DTCs in 163 pts with primary breast cancer, diagnosed between 2001 and 2004.
Patients and Methods: Tissue microarrays (TMA) were stamped from paraffin embedded specimens and 5µm thick sections were subjected to immunohistochemistry using antibodies against CD3 (DCS, Hamburg, Germany), CD68, CD20, CD8 (all Dakocytomation, Glostrup, Denmark), CD4 (Zytomed Systems, Berlin, Germany) and CD45R0 (Labvision, Fremont, USA), respectively. Each TMA has been evaluated for TILs and SILs assessing intraepithelial and stromal tumor areas occupied by respective immune cells separately: score 0: no staining; score 1: ≤ 10%; score 2: 11-60%; score 3: ≥61%, respectively, using light microscopy at magnifications of x100-200. Statistical analysis was conducted with the R programming language (version 3.20) and Cox proportional hazard regression and the Kruskal-Wallis test. DTCs were studied at the time of primary diagnosis using immunocytochemistry, applying the pan cytokeratin (CK) antibody A45-B/B3.
Results: Univariate analyses showed significant associations between prolonged OS and high rates of CD8TIL (p=0.01) and CD4TIL (p=0.028). In the subgroup of triple-negative carcinomas, CD3 TIL and CD8SIL were positively correlated with OS (p=0.038 and p=0.046, respectively), whereas CD8TIL was predictive in HR+/HER2- tumors (p=0.029). In a multivariate model, only CD4TIL was significantly associated with longer OS (p=0.049) in the total group of 163 pts, whereas in the triple-negative subgroup, CD3TIL was significantly correlated with favorable outcome (p=0.044). In contrast, no significant associations were found between TILs/SILs and PFS. The results for DTCs were available for 92/163 pts resulting in a positivity rate of 53%. Intriguingly, despite failing overall associations between DTCs and TILs/SILs, an unfavorable CD4SIL/CD8SIL ratio in the tumors of DTC-positive pts predicted significantly shorter PFS and OS (p=0.009 and p=0.018, respectively). In the DTC-negative subgroup, high CD4SILs were a prognostic marker for shorter OS. Notably, no significant associations were found between CD20, CD45R0 and CD68 and any parameter tested.
Conclusion: Analyzing a variety of leukocyte subtypes, we here demonstrate that especially the distribution of CD4 and CD8 in the primary tumor and their ratio seems to be involved in the prognosis of DTC-positive as well as DTC-negative primary breast carcinomas. Since the results for DTCs were only available in a subgroup of pts, further studies will have to prove these findings.
Citation Format: Kasimir-Bauer S, Spiekermann A, Friedrich CM, Hoffmann O, Schmid KW, Kimmig R, Bankfalvi A. Association between tumor infiltrating immune cells and disseminated tumor cells in the bone marrow of patients with primary breast cancer: A ten year follow-up. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-07.