Abstract P2-01-06: Similarities and differences in RNA profiles of circulating tumor cells in breast cancer subtypes: Do we have therapeutic options?
Authors: Bittner A-K , Hoffmann O , Keup C , Hauch S , Kimmig R , Kasimir-Bauer S 
Published: February 2018

Background: The success or failure of anti-cancer therapies in breast cancer (BC) is only assessed retrospectively by the absence or presence of overt metastases during the post-operative follow-up period. Circulating Tumor Cells (CTCs), the precursor of metastatic disease, would be an ideal surrogate tissue to identify prognostic and predictive factors directly at primary diagnosis guiding to optimal individual therapeutic strategies for metastasis prevention. Since the prognosis in BC has been shown to be different in various BC subtypes, CTCs present in each subtype may, therefore, also represent distinct metabolic profiles for survival, metastatic spread and therapy resistance Using a multi-marker gene panel for the characterization of the heterogeneous CTC population, we here compared the genetic profiles of CTCs in triple negative BC (TNBC) patients (pts) with CTC characteristics in non-TNBC pts.

Methods: 2x5 ml blood of 43 TNBC pts and 51 non TNBC pts (11= HER2+/HR-, 40=HER2-/HR+) before and/or after neoadjuvant therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)].

Results: PI3K and mTOR as well as the resistance markers ERCC1 and AURKA were predominantly expressed in all BC subtypes, the latter especially after therapy. EGFR could not be detected in any BC subtype. In TNBC pts, all the different genes were expressed, probably representing the most heterogeneous CTC population. Interestingly, HER2/HER3+ CTCs were found before and after therapy in more than 20% of these pts and mTOR/PI3K expression were not reduced after therapy. In HER2+/HR- pts, ALK, AR, c-KIT, HER3, KRT5 were never detected whereas NOTCH, PARP1 and SRC1 seemed to be induced by therapy in about 30% of the pts. Notably, HER2+CTCs, initially detected in 50% of the pts, completely disappeared after therapy, most likely due to anti- HER2 targeted treatment which seemed to also markedly reduce initial PI3K/AKT/mTOR expression whereas resistant, AURKA+ CTCs were found in 40% of cases before and after therapy. The expression pattern in CTCs of HR+/HER2- pts was, although to a lower extend, similar to the profile detected in HER2+/HR- pts but compared better to ERCC1 based resistance induction in TNBC pts.

Conclusion: Although CTCs in TNBC pts and non-TNBC pts show different genetic profiles, the PI3K/AKT/mTOR pathway as well as resistance markers seem to be commonly expressed in CTCs of all BC subtypes. This knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future.

Citation Format: Bittner A-K, Hoffmann O, Keup C, Hauch S, Kimmig R, Kasimir-Bauer S. Similarities and differences in RNA profiles of circulating tumor cells in breast cancer subtypes: Do we have therapeutic options? [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-06.