Abstract P6-01-02: Exploring biomarkers of response to zoledronic acid in breast cancer from clinical trial result of neoadjuvant chemotherapy with zoledronic acid: JONIE-1 study
Authors: Sangai Takafumi , Ishikawa Takashi , Kohno Norio , Miura Daishu , Sato Eiichi , Kaise Hiroshi , Suzuki Masato , Hasegawa Yoshie , Tanino Hirokazu , Horiguchi Jun , Akazawa Kohei 
Published: May 2015

Abstract
Background: Preclinical and clinical data have shown that zoledronic acid (ZOL) increases antitumor effect of chemotherapy (CT) in breast cancer (BC). We previously reported that the addition of ZOL to neoadjuvant CT is potentially beneficial in postmenopausal patients with triple-negative BC in JONIE-1 Study (0% pCR rate in CT versus 50% in CT combing ZOL, p=0.029). Our data suggest that there might be subpopulation that responds to ZOL addition; however, there is lack of evidence to explain mechanisms, emphasizing the need for exploring biomarkers of response using clinical samples. We hypothesized three mechanisms of antitumor effect of ZOL.
1. ZOL inhibits Src signaling pathway directory in BC cell.
2. ZOL suppresses releasing Insulin-like growth factor-1 (IGF-1) from the bone and decreases chemoresistance in BC.
3. ZOL alters immune response to BC especially through TAM which induces cytotoxic T cell infiltration.
Patients and Methods: We investigated the relationship between clinicopathological features and tumor shrinkage in 178 Stage IIA-IIIB HER-2-negative BC patients from the JONIE-1 adjuvant phase III trial comparing CT (FEC100 q3w × 4 cycles followed by weekly paclitaxel for 12 cycles) versus CT combining ZOL (4mg q3-4w). To evaluate Src activation, IGF-1 receptor (IGF-1R) activation, M1/ M2 macrophage infiltration, and cytotoxic T cells infiltration, we performed immunohistochemistry and two pathologists were independently performed assessment. Formalin fixed, paraffin embedded core needle biopsy sample at diagnosis and surgical specimen after neoadjuvant CT were serially sectioned at 4 micrometer and processed for HE staining and immunohistochemistry using primary antibodies as follows; Rabbit anti-Src mAb (36D10, Cell signaling), Rabbit polyclonal anti-p-IGF-IR Ab (Tyr 1161) (Santa-Cruz Biotechnology), Mouse anti-CD80 mAb, (UMAB65, OriGene Technologies), Mouse anti-CD163 mAb (10D6, Lwica Biosystems), Mouse Anti-CD8 mAb (C8/144B, Dako).
Results: All immunohistochemistry were successfully performed. Src activation was recognized as peripheral membrane localization. Stained slides are now under evaluation by pathologist. Associations between clinicopathological features and the effect of CT with ZOL will be under investigation.
Discussion: Src activation was observed in more than 70% of triple negative BC. Multiple cellular functions of Src are mediated by Ras which is the main target of ZOL in osteoclasts. IGF-1R was overexpressed in approximately 50% of BC and IGF-1 signaling protects BC cell from CT by proliferative and anti-apoptotic effects. IGF-1 is the most released growth factor from the bone matrix during bone resorption and ZOL administration resulted in a significant decrease in IGF-1. Subclinical inflammation induced by macrophages in adipose tissue play an important role in promoting BC growth. It has been reported that ZOL reverted tumor-infiltrating macrophages (TAM) phenotype from M2 to M1. Therefore tumor infiltrating lymphocytes would be dominantly reverted to cytotoxic T cells for inhibiting tumor growth as well. We will present the results and discuss antitumor effects of ZOL at the meeting.
Citation Format: Takafumi Sangai, Takashi Ishikawa, Norio Kohno, Daishu Miura, Eiichi Sato, Hiroshi Kaise, Masato Suzuki, Yoshie Hasegawa, Hirokazu Tanino, Jun Horiguchi, Kohei Akazawa. Exploring biomarkers of response to zoledronic acid in breast cancer from clinical trial result of neoadjuvant chemotherapy with zoledronic acid: JONIE-1 study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-02.