Abstract P2-02-01: In primary breast cancer patients with HER2-negative tumors, HER2-positive circulating tumor cells with stem cell character predict worse outcome
Authors: Kasimir-Bauer S , Bittner A-K , Aktas B , Hauch S , Kimmig R , Hoffmann O 
Published: May 2016

Abstract
Introduction: The detection of disseminated (DTCs) and circulating (CTCs) tumor cells in patients with early stage breast cancer is a well described independent prognostic factor associated with increased risk of disease recurrence and disease-related death. We recently demonstrated that CTCs in blood of primary breast cancer patients rather than DTCs were significantly associated with reduced progression free survival (p=0.02). Using comprehensive molecular characterization, we here demonstrate that the negative prognostic impact was predominantly related to HER2-positive CTCs with stem cell character from patients with HER2-negative primary tumors.
Patients and Methods: 2 x 5 ml blood from 482 primary breast cancer patients with first diagnosis between 2006 and 2010 were analyzed for CTCs with the AdnaTest BreastCancer (QIAGEN Hannover GmbH, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was now additionally tested for the expression of the estrogen (ER) and progesterone receptor (PR) (single-plex RT-PCR) and stem cell like CTCs (slCTCs) applying the AdnaTest TumorStemCell (single-plex RT-PCR for ALDH1) and the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K). The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100.
Results: CTCs were detected in 103/482 (21%) of the patients expressing EpCAM (27%), MUC-1 (26%), HER-2 (75%), ER (14%) and PR (8%), respectively. Notably, in 49/103 (48%) of the CTC-positive patients, HER2 was the only marker expressed. slCTCs could be analyzed in 72/103 CTC-positive patients. At least one of the EMT markers was expressed in 56/72 patients (78%), ALDH1 was present in 32/72 patients (44%) and 31/72 (44%) were positive for both, ALDH1 and EMT markers, respectively. Comparisons of expression profiles on CTCs with those on the primary tumor were only performed in CTC-positive patients. Primary tumors and CTCs displayed a concordant HER2, ER and PR status in 37% (p=0.81), 24% (p=0.257) and 27% (p=0.876) of cases, respectively. Most interestingly, in 60/75 (80%) patients with HER2-positive CTCs, primary tumors were HER2-negative. In contrast, the percentage of patients with ER- and PR-positive CTCs but negative ER/PR primary tumors was 29% and 25%, respectively. When the presence of HER2-positive CTCs was correlated with the presence of slCTCs (ALDH1-and/or EMT-positive), the concordance was 83% (p=0.0019). In detail, the concordances were HER2+ vs ALDH1+ (54%, p=0.037), HER2+ vs EMT+ (85%, p=0.0002) and HER2+ vs ALDH1+/EMT+ (80% p=0.00053), respectively.
Conclusion: Our results provide evidence that a) the negative prognostic impact of CTCs in our patient cohort is related to HER2-positive CTCs with EMT and tumor stem cell characteristics which indicate therapy resistant tumor cell populations and, therefore, an inferior prognosis and b) “secondary” adjuvant treatment with HER2-targeting agents, alone or in combination, may probably be effective to eliminate these cells and thus, lead to an overall decreased relapse rate. This study further confirms that the molecular characterization of CTCs might help to stratify patients for individual treatment options.
Citation Format: Kasimir-Bauer S, Bittner A-K, Aktas B, Hauch S, Kimmig R, Hoffmann O. In primary breast cancer patients with HER2-negative tumors, HER2-positive circulating tumor cells with stem cell character predict worse outcome. [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 P2-02-01.