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Patient characteristics of the whole breast cancer cohort used for CX3CL1 expression analysis (n=250)

CX3CL1 mRNA expression is associated with improved recurrence-free, distant-metastasis free, and overall survival in human breast cancer.

Figure S2. Impact of Cx3cl1 overexpression on tumor cell growth in vitro, Cx3cl1 serum concentrations, and myeloid cell tumor infiltration in the 4T1 breast cancer model.

Impact of Cx3cl1 overexpression in the HT-29 xenograft model.

Impact of soluble CX3CL1 and ADAM17 inhibition by TAPI-2 on NK cell-mediated tumor cell lysis in vitro.

Impact of Cx3cl1 overexpression in the MDA-MB 453 xenograft model.

Modulation of CXCL10 secretion from human HER2-positive cancer cell lines upon stimulation with TNF-α and TAPI-2.

A crucial mode of action of trastuzumab is the labeling of HER2-positive (HER2+) tumor cells for the eradication by natural killer (NK) cells, a process called antibody-dependent cellular cytotoxicity (ADCC). However, despite widespread HER2 expression among cancer entities, only a fraction, with robust HER2 overexpression, benefits from trastuzumab therapy. ADCC requires both sufficient lymphocytic infiltration and close binding of the immune cells to the antibody-tagged tumor cells. We hypothesized that the chemokine CX3CL1 could improve both processes, as it is synthesized as a membrane-bound, adhesive form that is eventually cleaved into a soluble, chemotactic protein. Here, we show that CX3CL1 overexpression is a positive prognostic marker in breast cancer. CX3CL1 overexpression attracted tumor-suppressive lymphocytes, including NK cells, and inhibited tumor growth and lung metastasis in the syngeneic 4T1 breast cancer mouse model. In HER2+ SKBR3, MDA-MB-453, and HT-29 tumor cells, CX3CL1 overexpression increased NK cell–mediated cytotoxicity in vitro and acted synergistically with trastuzumab. Even though CX3CL1 did not further improve trastuzumab efficacy in vivo in the trastuzumab-sensitive MDA-MB-453 model, it compensated for NK-cell depletion and prolonged survival. In the HER2 low–expressing HT-29 model, however, CX3CL1 overexpression not only prolonged survival time but also overcame trastuzumab resistance in a partly NK cell–dependent manner. Taken together, these findings identify CX3CL1 as a feasible pharmacologic target to enable trastuzumab therapy in HER2 low–expressing cancers and render it a potential predictive biomarker to determine therapy responders.

CX3CL1 overexpression in HER2 overexpressing human breast cancer.

Background: The Endopredict test is used for estimating risk of distant recurrence for women presenting with early-stage breast cancer with a positive estrogen receptor (ER) and negative human epidermal growth factor receptor 2 (HER2) status. The current ASCO Guideline Update on biomarkers confirms the value of the Endopredict test to guide decisions of adjuvant endocrine and chemotherapy. This study shows prospective long-term outcome data of early breast cancer patients whose chemotherapy decision was guided by the Endopredict test result (EPclin). Methods: ER-positive and HER2-negative early breast cancer patients with 0-3 positive lymph nodes treated between March 2012 and March 2015 were included in this single institution study. The Endopredict® test was carried out on all tumour samples. Demographic, clinical and pathological data were assessed for each patient at baseline. Treatment compliance, local recurrence, distant metastases and survival was evaluated. Risk estimates were obtained by the Kaplan-Meier method and cumulative risk functions in case of competing risks. Group comparisons were performed by Cox proportional hazards regression models and quantified through hazard ratios. Median Follow-Up was estimated by the inverse Kaplan-Meier method. Exploratory hypothesis testing was conducted at two-sided 5% significance levels. Results: In a cohort of 368 consecutive cases the median follow-up time was 8.2 years. Endopredict allocated 238 pts (64%) in the EPclin low risk and 130 pts (34%) in the EPclin high risk group. The 5-year distant metastasis free survival (DMFS) in the EPclin low risk group was 96.6% (95% CI 0.943-0.989) and 85.5% (95% CI 0.796-0.920) in the EPclin high risk group. With a hazard ratio (HR) of 2.21 (95% CI: 1.27-3.88; p=0.005) the risk for distant metastasis in EPclin high risk patients was more than two-fold higher in comparison with EPclin low risk patients. 87 pts. (66.9%) of the EPclin high risk group underwent chemotherapy (compliant), whereas 43 pts (33.1 %) opposed the recommended chemotherapy (non-compliant). Kaplan-Meier plots in the EPclin high risk subgroups compliant vs non-compliant showed a significant disease-free survival (DFS) benefit towards the patients following the chemotherapy recommendation (HR 0.46; 95%CI 0.23-0.95; p=0.036). The 5-year DFS for the high risk compliant subgroup was 89.1% (95% CI: 0.827-0.961) vs. the high risk non-compliant subgroup with 68.9% (95% CI: 0.562-0.845). Regarding the subgroups pre- and postmenopausal, patients with a EPclin high risk test result were at significant higher risk of experiencing distant metastases than patients with a EPclin low risk test result in both subgroups (premenopausal: HR 3.55; 95%CI 1.17-12.32; p=0.025; postmenopausal: HR 1.19; 95%CI 0.99-3.7; p=0.054). We analyzed the EPclin categorization in context of the ki67 subtypes luminal A (low; 0-10%) and luminal B (high; 25-100%). The EPclin-based risk stratification was significantly associated with improved DFS in both ki67 subtypes (ki67 low: HR 4; 95%CI 1.25-12.04; p=0.021 and ki67 high: HR 3.77; 95%CI 1.19-18.93; p=0.022). 33.3% (21 pts) of all tumor samples classified as luminal B (63 pts), were reclassified towards the low risk group via Endopredict, sparing chemotherapy recommendation. Contrary 19.2% (14 pts) of all luminal A (73 pts) were categorized to high risk EPclin. Conclusion: These first long term prospective outcome data confirm, that Endopredict can guide decisions on adjuvant chemotherapy in early ER positive, HER2 negative breast cancer. Pts categorized as EPclin high risk benefitted from an adjuvant chemotherapy. Our results show that Endopredict risk stratification is also applicable in premenopausal women. Furthermore the Endopredict test showed a better classification accuracy in comparison to ki67 subtypes, resulting in a more precise estimation of prognosis. Citation Format: Evelyn Klein, Adriana Josipovic, Aurelia Noske, Sophie Anders, Carolin Mogler, Wilko Weichert, Alexander Hapfelmeier, Marion Kiechle, Johannes Ettl. Long-term outcome data using Endopredict® as risk stratification and chemotherapy decision biomarker in hormone receptor positive, HER2-negative early breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-24.

Background: The EndoPredict test is a clinico-molecular test that has been validated to predict the likelihood of distant metastases and late relapse inpatients (pts) with hormone receptor positive (HR+), HER2-negative (HER2-) early breast cancer and up to three positive lymph nodes. However, so far prospective outcome results of pts in whom decision on use of adjuvant chemotherapy (CTX) was based on Endopredict has not been available. Here we present three year outcome data of pts, whose adjuvant systemic therapy recommendation was based on EndoPredict test result. Methods: Pts with HR+, HER2- early breast cancer with 0-3 positive lymph nodes were enrolled at a single institution (Interdisciplinary Breast Center of Klinikum rechts der Isar, Munich, Germany). The Endopredict test was carried out on all tumor samples. Demographic, clinical and pathological data as well as EPclin risk class were assessed for each patient at baseline. Therapy recommendations were given during an interdisciplinary tumor board. Pts were evaluated for treatment compliance, local recurrence, distant metastases and survival (cut-off date of last follow up: July 31st 2017). Censored time-to-event outcomes were analysed by cox proportional hazards models. Additional estimates of the event-free-survival were given by the Kaplan-Meier method. Hypothesis testing was conducted on two-sided exploratory 5% significance levels. Results: A total of 373 consecutive pts were enrolled between March 2012 and March 2015. Median age was 59.9 (range: 29.1-88.9) years. In 39% of pts tumorsize was >2cm, 24% of pts were node positive, 16% had G3 tumors, in 21% of pts ki67 was ≥25% and in 13% progesterone receptor was Conclusion: These first prospective outcome results show, that EPclin in clinical routine is a valid clinico-molecular marker to predict DFS and guide decision of adjuvant CTX use in HR+, HER2- early breast cancer pts with 0-3 positive lymph nodes. Citation Format: Ettl J, Anders S, Hapfelmeier A, Noske A, Paepke S, Weichert W, Klein E, Kiechle M. First prospective outcome data for the clinico-molecular test Endopredict® in hormone receptor positive, HER2-negative early breast cancer in clinical routine [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-11.

Introduction: Based on high-throughput transcriptional profiling and array comparative genomic hybridization, a three-gene predictor encompassing c-MYC, EGFR and FGFR2 has been identified to predict survival in cisplatin and fluorouracil (FU) – treated metastatic gastric cancer (GC) patients by others (1). The aim of our study was to determine the value of the expression of these three genes to predict response and/or survival for GC patients treated by a platin/5FU based chemotherapy in the neoadjuvant setting. Material and methods: Expression was analyzed in pretherapeutic tumor biopsies of 69 patients treated by platinum/5FU based neoadjuvant chemotherapy. Histopathological response was divided into three grades, tumor regression grade 1 (TRG1) corresponding to complete/major response, TRG2 corresponding to partial and TRG3 corresponding to minimal or no response. mRNA was isolated from manually microdissected formalin fixed and paraffin embedded tumor biopsies. Relative quantification of gene expression was performed by real time PCR using the delta-delta Ct method. As reference genes IPO8 and POLR2A were used and normalization was based on geometric averaging. Analyses were done in triplicates and the mean value was calculated. Fisher`s exact test or χ2-test (two sided) were used to test for an association with response. Overall survival was analyzed by log rank tests and Cox regression analyses. Results: Performing dichotomization of the patients based on median gene expression levels revealed a significant association of high c-MYC expression with minimal or no response (TRG3) (p=0.013). No other associations with response were observed for EGFR or FGFR2 expression in the study as a whole or in a separate analysis of patients with intestinal and nonintestinal type tumors. Testing various combinations of the expression of the three genes did not reveal a stronger association with response when compared to c-MYC expression alone. In the study as a whole, no statistically significant associations with overall survival were found. In the subgroup of nonintestinal-type tumors, univariate Cox regression analysis revealed a significantly increased risk of death for patients with a high expression of c-MYC (HR 59.56, 95%CI 1.28-2776.3; p=0.037). Conclusion: Our results indicate a prominent role of c-MYC expression to predict tumor shrinkage after platin/5FU-based neoadjuvant chemotherapy in GC and they suggest a prognostic relevance of this gene in particular for patients with nonintestinal-type gastric carcinomas. Reference: 1. Kim HK et al.: The Pharmacogenomics Journal (2012) 12: 119-127. Citation Format: Anna Munzig, Lukas Bauer, Julia Slotta-Huspenina, Alexander Novotny, Karen Becker, Alexander Hapfelmeier, Gisela Keller. c-MYC, EGFR, and FGFR2 expression and response and survival in neoadjuvant treated gastric cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 12.