Purpose Phosphatidylinositide-3-kinase (PI3K) regulates proliferation and apoptosis; somatic PIK3CA-mutations may activate these processes. Aim of this study was to determine the prevalence of PIK3CA-mutations in a cohort of early stage breast cancer patients and the association to the course of disease. Patients and methods From an unselected cohort of 1270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825) 1123 tumours were tested for the three PIK3CA hotspot-mutations H1047R, E545K, and E542K by qPCR. Primary objectives were the prevalence of somatic PIK3CA-mutations and their association to tumour characteristics. Secondary objective was the association of PIK3CA-mutations to recurrence-free interval (RFI) and overall survival. Results PIK3CA-mutation rate was 26.7% (300 of 1123). PIK3CA-mutations were significantly more frequent in steroid hormone-receptor (SHR)-positive HER2-negative (31.4%), and G1 and G2 tumours (32.8%). Overall, we did not observe a significant association of PIK3CA-mutations to RFI. In SHR-positive BCs with PIK3CA-mutations, a strong trend for impaired  RFI was observed (adjusted HR 1.64, 95% CI 0.958–2.807), whilst in SHR-negative BCs PIK3CA-mutations were insignificantly associated with improved RFI (adjusted HR 0.49; 95% CI 0.152–1.597). Of note, we observed a significantly detrimental prognostic impact of PIK3CA-mutations on RFI in SHR-positive, HER2-negative BCs if only aromatase inhibitors were administered as adjuvant therapy (adjusted HR 4.44, 95% CI 1.385–13.920), whilst no impact was observed in tamoxifen treated patients. Conclusion This cohort study speficies the overall mutation rate of PIK3CA in early breast cancer. The impact of PIK3CA-mutations on RFI and OS was heterogeneous. Our results suggest that estrogen deprivation failes to be active in case of PIK3CA-mutation.

<b><i>Introduction:</i></b> Triple-negative breast cancer (TNBC) is considered the most aggressive type of breast cancer (BC) with limited options for therapy. TNBC is a heterogeneous disease and tumors have been classified into TNBC subtypes using gene expression profiling to distinguish basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor (LAR), and one nonclassifiable group (called unstable). <b><i>Objectives:</i></b> The aim of this study was to verify the clinical relevance of molecular subtyping of TNBCs to improve the individual indication of systemic therapy. <b><i>Patients and Methods:</i></b> Molecular subtyping was performed in 124 (82%) of 152 TNBC tumors that were obtained from a prospective, multicenter cohort including 1,270 histopathologically confirmed invasive, nonmetastatic BCs (NCT 01592825). Treatment was guideline-based. TNBC subtypes were correlated with recurrence-free interval (RFI) and overall survival (OS) after 5 years of observation. <b><i>Results:</i></b> Using PAM50 analysis, 87% of the tumors were typed as basal with an inferior clinical outcome compared to patients with nonbasal tumors. Using the TNBCtype-6 classifier, we identified 23 (15%) of TNBCs as LAR subtype. After standard adjuvant or neoadjuvant chemotherapy, patients with LAR subtype showed the most events for 5-year RFI (66.7 vs. 80.6%) and the poorest probability of 5-year OS (60.0 vs. 84.4%) compared to patients with non-LAR disease (RFI: adjusted hazard ratio [aHR] = 1.87, 95% confidence interval [CI] 0.69–5.05, <i>p</i> = 0.211; OS: aHR = 2.74, 95% CI 1.06–7.10, <i>p</i> = 0.037). <b><i>Conclusion:</i></b> Molecular analysis and subtyping of TNBC may be relevant to identify patients with LAR subtype. These cancers seem to be less sensitive to conventional chemotherapy, and new treatment options, including androgen receptor-blocking agents and immune checkpoint inhibitors, have to be explored.

Tumour-infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. We examined 1166 breast cancer patients from a prospective, multicentre cohort (Prognostic Assessment in Routine Application (PiA), n = 1270, NCT 01592825) following recommendations from the International TILs Working Group. TIL quantification was performed using predefined groups and as a continuous variable in 10% increments. The primary objective was the distribution of TILs in different breast cancer types. The second objective was the association with the recurrence-free interval (RFI) and overall survival (OS). Stromal infiltration with more than 60% TILs appeared in 2% of hormone receptor (HR)-positive and HER2-negative tumours, in 9.8% of HER2-positive tumours (any HR) and 19.4% of triple-negative breast cancers (TNBCs). Each 10% increment was associated with an improvement in the prognosis in HER2-positive samples (RFI, hazard ratio 0.773, 95% CI 0.587–1.017; OS, hazard ratio 0.700, 95% CI 0.523–0.937). When defining exploratory cut-offs for TILs, the use of a 30% threshold for the HR-positive and HER2-negative group, a 20% threshold for the HER2 group and a 60% threshold for the TNBC group appeared to be the most suitable. TILs bore prognostic value, especially in HER2-positive breast cancer. For clinical use, additional research on the components of immune infiltration might be reasonable.

Introduction: Triple negative breast cancer (TNBC) is, in general, associated with unfavorable prognosis. However, TNBC it is a heterogenous disease. Thus, we analyzed the prognosis (RFI, OS) of TNBC in association to the molecular subtypes (according to Lehmann et al. 2011) and the prognostic markers uPA (urokinase-type plasminogen activator) and its inhibitor (uPA/PAI-1) within a prospective cohort of breast cancer patients. Material & Methods: The PiA-study was designed as a multicenter cohort of early BC patients (n=1270, 2009 – 2011), treated according to national guidelines from six centers in Germany (Prognostic assessment in routine Application, NCT 01592825). All 153 patients with TNBC (12%) received chemotherapy (neoadjuvant, n=99, or adjuvant, n=54). Tumor characteristics were based on local pathology. IN 81 TNBC patients, fresh frozen tissue was obtained for central uPA/PAI-1 testing by ELISA (FEMTELLE BioMedica Diagnostic) was available. RNA of formalin fixed and paraffin embedded (FFPE) TNBC tumor material (n=125) was analyzed using the GeneChip® U133 Plus 2.0 (Affymetrix). The online Vanderbilt predictor tool was used to determine molecular subtypes (http://cbc.mc.vanderbilt.edu/tnbc). Results: In the total cohort, 89.5% (CI 87.7-91.3) were free of RFI events after 5 years, the TNBC patients showed the poorest outcome with 72.8% (CI 65.5-80.1). In TNBC patients with a low uPA/PAI-1 status significantly more were free of RFI events than in patients with high uPA/PAI-1 status: 93% (CI 79.4-106.4) and 70.5% (CI 58.7-82.3), respectively. By the type 6-Vanderbilt tool, molecular subtypes were assigned as follows: BL1 18%, BL2 12%, IM 22%, M 12% MSL 6%, LAR: 14%, and UNS 16%. Using the type 4 tool we found for BL1 30%, BL2 18%, M 15%, LAR 18%, and UNS 19%. In all survival analysis, the patients with LAR subtype (n=23) had the lowest survival probability, with BL2 subtype N=22) the best: for 5 year RFI 58.7% (CI 29.4-71.8) versus 81.6% (CI 65.3-97.9). In those 54 TNBC patients who were treated with neoadjuvant chemotherapy, 46% of the patients had a pathologically confirmed complete response (ypT0/is ypN0). Counclusion: TNBC is a heterogeneous disease. Patients with TNBC and low uPA/PAI-1 expression have a very low rate of disease recurrence. Particular attention has to be drawn to patients with the LAR subtype, since it seems to have the worst prognosis and may require androgen receptor directed treatment. Outcome results of different endpointsn=125BL1 (n=38)BL2 (n=22)M (n=18)LAR (n=23)UNS (n=24)OS (95% CI)80.3% (67.2-93.4)95.2% (86.2-104.2)71.8% (50.8-92.8)65.2% (44.4-84.4)78.4% (61.5-95.3)RFS (95% CI)78.1% (64.6-91.6)81.6% (65.3-97.9)66.7% (44.9-88.5)50.6% (29.4-71.8)78.6% (62-95.2)RFI (95% CI)78.1% (64.6-91.6)81.6% (65.3-97.9)66.7% (44.9-88.5)58.7% (37.3-80.1)91.3% (79.7-102.9)BCSS (95% CI)80.3% (67.2-93.4)95.2% (86.2-104.2)71.8% (50.8-92.8)82.4% (66.7-98.1)90.9% (78.9-102.9) Citation Format: Martina Vetter, Carolin Hartung, Tilmann Lantzsch, Volker Hanf, Christoph Uleer, Susanne Peschel, Jutta John, Joerg Buchmann, Edith Weigert, Karl-Friedrich Buerrig, Eva-Johanna Kantelhardt, Christoph Thomssen. Prospective analysis of molecular subtypes in triple negative breast cancer [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 P4-06-13.

Introduction The PiA-study (Prognostic assessment in routine Application, NCT 01592825) was designed as a representative cohort of breast cancer patients to estimate the proportions of traditional and modern prognostic factors. The ASCO-recommended biomarkers uPA (urokinase-type plasminogen activator) and its inhibitor PAI-1 were used for biological risk assessment particularly for intermediate risk breast cancer patients and disease-free survival of the patients after 5 yrs of follow-up (F/U) was calculated. Material & Methods Between 2009 and 2011, 1,074 non-metastasized, primarily operated breast cancer patients from six centers in Germany were included. From 815 patients, fresh frozen tissue was obtained and processed for central testing uPA/PAI-1 by ELISA (FEMTELLE®, Sekisui Diagnostics GmbH). Low uPA/PAI-1 status is defined by uPA and PAI-1 concentrations below the published cut-offs, high status means one or both were higher than the corresponding cut-offs. Tumor characteristics were based on local pathology. The centers had to follow the national guidelines. In low-risk patients, adjuvant chemotherapy was spared. The median F/U is 56 months (range 0-78). Results In the total cohort of 1,074 patients, 166 had G1- and 237 had G3-tumors. Of the 671 patients with a G2-tumor, the following were allocated to the high-risk group: node-positive (n=371), younger than 35 yrs (n=17), and triple-negative (TN) or HER2-positive (n=118). For 253 tumors of the remaining 355 patients with an intermediate risk of recurrence (pN0, G2, HR positive, HER2-negative, ≥35 yrs), uPA/PAI-1 status was available. 126 (49.8%) were allocated to the low-risk group, one patient had a recurrence. At 5 yrs, in the total cohort 90.6% (95% CI, 89.5-91.7) of the patients were free of invasive disease. Of 114 HER2-positive tumors, 94 (82.4 %) had a high uPA/PAI-1 status, only one of the 38 HR negative/HER2 positive tumors had a low uPA/PAI-1 status. In the TN group, the majority of tumors had a high uPA/PAI-1 status (66 of 81; 81.5 %). In 30 patients lymph nodes were involved, 18.5% (n=15) had a low uPA/PAI-1 status, one event was detected. In N pos. patients with an high uPA/PAI-1 6 events were observed. Conclusion Testing for uPA/PAI-1 in the daily routine is feasible, fresh frozen tissue has been prepared from 76% of the tumors of the recruited patients, 37% of them had a low risk status. Using uPA/PAI-1, about half of the node-negative patients with an intermediate risk of recurrence were allocated to a group with an extremely low risk of recurrence and thus chemotherapy could be spared. Also in node-positive disease, uPA/PAI-1 has a prognostic impact. Tab 1: Proportion of the subgroups according to IHC, grading and uPA/PAI-1-statusTumor typetotallow uPA/PAI-1 statushigh uPA/PAI-1 statusn=815 (100%)n=304 (37%)n=511 (63%)Luminal A-like tumors:HRpos., HER2neg., G1, G2515 (63.2%)240 (78.9%)275 (53.9%)Luminal B/HER2-negative-like tumors:HRpos., HER2neg., G3104 (12.8%)29 (9.5%)75 (14.7%)Luminal B/HER2-positive-like tumors:HR pos., HER2 pos., all grades77 (9.4%)19 (6.2%)58 (11.4%)HER2-positive (nonluminal) - like tumors:HRneg., HER2pos., all grades38 (4.7%)1 (0.3%)37 (7.3%)TN tumors:HRneg., HER2neg., all grades81 (9.9%)15 (4.9%)66 (12.9%) Citation Format: Vetter M, Hartung C, Hanf V, Lantzsch T, Uleer C, Peschel S, John J, Buchmann J, Bürrig K-F, Weigert E, Thomssen C, Kantelhardt EJ. The ASCO-recommended prognostic factors uPA/PAI-1 in a multicenter cohort study (PiA) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-12.