Abstract 369: Association between tumor infiltrating immune cells, circulating tumor cells in blood and disseminated tumor cells in the bone marrow in patients with primary ovarian cancer
Authors: Chebouti Issam , Bankfalvi Agnes , Friedrich Christoph , Kimmig Rainer , Kasimir-Bauer Sabine 
Published: August 2015

Abstract
Background: We recently showed that the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) and circulating tumor cells (CTCs) in blood of primary ovarian cancer patients significantly correlated with reduced progression free survival (PFS) and overall survival (OS). On the one hand, this negative prognostic impact might be related to different factors like chemotherapy resistant and stem cell like cells. On the other hand, the microenvironment of the primary tumor might influence tumor cell spread and thus, outcome of the disease. Here we analyzed whether infiltrating leukocytes in the tumor (TILs) and/or stroma (SILs) are associated with DTCs before and after therapy as well as with CTCs before therapy in patients with primary ovarian cancer.
Patients and Methods: DTCs before (n = 145) and after therapy (n = 57) were studied using immunocytochemistry applying the pan cytokeratin (CK) antibody A45-B/B3. 2 × 5 ml bloods of 111 patients were analyzed for CTCs with the AdnaTest OvarianCancer (AdnaGen AG, Langenhagen, Germany) for the detection of EpCAM, MUC-1, CA-125 and beta-Actin transcripts. Tissue microarrays of all patients were stamped from paraffin embedded specimens and 5μm thick sections were prepared and subjected to immunohistochemical analysis using antibodies against human CD4 (Zytomed Systems, Clone IF6, 1:40), CD8 (DakoCytomation, Clone C8/144B, 1:150) and CD68 (DakoCytomation,Clone PG-M1, 1:500), respectively. Each tissue microarray has been evaluated for TILs and SILs by counting manually, using a 10x and 20x objective lens. Statistical analysis was conducted with the R programming language (version 3.02) and ANOVA, Chi-squared test, and when indicated, Fisher's exact test has been used to check for independence.
Results: Before therapy, DTCs were detected in 51/145 patients (35%) and after therapy in 24/57 patients (42%). CTCs were detected in 24/111 patients (22%) before therapy. In general, the highest infiltration of lymphocytes was found in the stroma. Whereas high numbers of CD8 were detected in the stroma and the tumor, high numbers of CD4 and CD68 infiltrates were mainly found in the stroma. The expression of CD4 (SIL) and CD8 (SIL) was significantly associated with the presence of CTCs (p = 0.04) whereas CD4 (SIL) significantly correlated with DTCs before (p = 0.03) and CD8 (SIL) with DTCs after therapy (p = 0.04). No association was found between DTCs/CTCs and the expression of CD68.
Conclusion: Here we demonstrate significant associations between infiltrating lymphocytes and the presence of DTCs and CTCs. Ongoing studies are now including the expression of CD20 and CD45 and final statistical analysis, including immune signatures, all clinical parameters, PFS and OS, will give deeper insights in the interaction of tumor infiltrating immune cells and tumor cell dissemination.
Citation Format: Issam Chebouti, Agnes Bankfalvi, Christoph Friedrich, Rainer Kimmig, Sabine Kasimir-Bauer. Association between tumor infiltrating immune cells, circulating tumor cells in blood and disseminated tumor cells in the bone marrow in patients with primary ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 369. doi:10.1158/1538-7445.AM2015-369