M Kusche

Abstract P6-05-11: Run-in phase of prospective WSG-ADAPT HR+/HER2- trial demonstrates feasibility of early endocrine sensitivity prediction by recurrence score and conventional parameters in clinical routine

Background: Despite promising evidence regarding outcome prediction, endocrine sensitivity, as determined by proliferation response to short-term preoperative endocrine therapy, is currently not included in adjuvant chemotherapy decisions in early HR+/HER2- breast cancer (BC). Methods: The prospective WSG-ADAPT HR+/HER2- trial includes early BC patients with 0-3 positive LN who are candidates for adjuvant chemotherapy based on clinical-pathological criteria alone; it aims to spare chemotherapy in a substantial proportion utilizing a combination of genomic assessment by Oncotype DX and endocrine sensitivity testing. All patients received 3-week preoperative endocrine induction therapy (ET): aromatase inhibitors (AI) if postmenopausal, tamoxifen if premenopausal. Patients with low (0-11) Recurrence Score (RS) or intermediate RS (12-25) and ET response (centrally tested, post-therapy Ki-67 Results: As of 6/2013, 380 patients from 30 study centers had been enrolled in the ADAPT HR+/HER2- trial. Median age was 54 years. At first pre-planned analysis (5/2013), paired Ki-67 measurements (pre-/post-therapy) were available in 241 patients; RS was available in 208 cases (201 with paired Ki-67). RS was low in 21.6%, intermediate in 57.7%, and high in 20.7%; the respective risk group responder percentages (post-treatment Ki 67 70% endocrine responders in the intermediate genomic risk group, who could potentially be spared adjuvant chemotherapy. Median Ki 67 level decreases (as percentage of pre-treatment value) were 25% in premenopausal patients (tamoxifen, n = 101) vs. 75% in postmenopausal patients (AI, n = 115) (p Conclusions: The Run-In Phase of the WSG ADAPT HR+/HER2- trial confirms trial design estimates of RS and proliferation response to induction ET. It indicates that the multicenter prospective ADAPT concept combining static and dynamic biomarker assessment for individualized therapy decisions in early BC is feasible. Proliferation response was strongly associated with therapy group (AI/post-menopausal vs. tamoxifen/pre-menopausal). Survival non-inferiority of intermediate Recurrence Score proliferation responders vs. low Recurrence Score patients (active control) will be tested in the ADAPT main phase to determine if adjuvant chemotherapy can be spared in 70% of patients with 0-3 positive LN classified as “intermediate risk” by conventional factors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-11.

Abstract P1-04-09: Extended characterization and target analysis of circulating epithelial cells in early and late breast cancer

The MAINTRAC based (RBC lysis immunofluoretic detection and analysis on the Olympus ScanR system) finds more cells compared to enrichment based systems. The relative cheapness and low amounts of blood (1ml) allows frequent monitoring during early and late disease therapy steps. The clinical relevance of rising or falling counts of living (EPCAM+CD45negDAPIneg) CEC has been demonstrated in early and late disease by Pachmann. In addition we could show that these cells express stem cell, EMT and hypoxia markers. To our surprise we found high counts of this cell type in 90% of patients with fatty liver disease (NAFLD) in a non healthy non malignant control population. In a healthy control population in only 2% very few cells were found. Our conclusion so far CEC are a phenomenon driven by a hypoxic hyperacidic tissue situation shared by malignant and non malignant disease. The detectable cell population showed considerable morphologic heterogeneity urging us to introduce extended characterization and more comprehensive analysis. We therefore slightly adapted the basic MAINTRAC procedure to allow four color flowcytometric detection and analysis on the Amnis (EMD-Millipore) FlowSight device. The basic panel consists of (CD45PE, DAPI- blue, ALDH-FITC and EPCAMAF660). Extended Characterization is possible with different other panels including (HIF1, betacatenin, several pan- and single cytokeratins and CD44/CD24) and targets like ER, HER2, PDL-1 (immune checkpoint target) and phosphatidylserine. With this new approach we examined so far 66 patients with early and 53 patients with late disease. In early disease we found living CEC in 32 of 66 cases, with cell counts from 200 to 21642 per ml. In late disease in 27of 53 cases, cell counts varying from 481 to 97339 cells per ml. The percentage of living CEC varied from 0 to 60%. In all cases we found PDL-1 and ALDH1 expressed nearly exclusively on EPCAM+ cells demonstrating their suitability as a CEC specific marker. In cases with early relapse and rapid progression only few or none CEC are found especially in triple negative and HER2+ER negative cases. In these situations we find however EPCAM negative cell clusters. The IDEAS software allows comprehensive analysis of all visible phenomena including single cells, cell aggregates and exosomes. So far we still see no differences between “real” tumor derived CEC and “false positive” liver derived CEC. There is however preliminary evidence that CK20+ cells counts are much higher than EPCAM +counts in real compared with liver caused disease. A conclusive picture of these intruiging phenomena is not possible it seems however that the chosen way of extended characterization including new issues like cell clusters and exosomes should lead to a further elucidation of this biology and the use of CEC as a reliable “fluid biopsy”. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-09.

Abstract P1-07-16: Liver derived epithelial cells as source of false positive circulating tumor cells in early breast cancer

The MAINTRAC technique as introduced by our coworkers from Jena (RBC lysis, fluorometric detection and analysis on Olympius ScanR) detects more circulating epithelial cells than techniques using enrichment. Also cells with a low EPCAM expression are detected and not only the typical cells with bright expression found after immunomagnetic enrichment. The relative cheapness and reproducibility allows frequent monitoring during and after therapy Using 3 colour detection (EPCAMfitc, DAPI, Vimentin PE) living and dead circulating epithelial cells in EMT, or cells in EMT with stemcell markers (EPCAMfitc, Vimentin-PE, CD44PacBlue) can be detected. In early breast cancer (n = 135) cells can be found in 60% of patients and in 40% higher cell counts (>100 ml are detectable. A control population(n = 100) showed low numbers in 98% (e.g ( Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-16.