GBCC 2017: ABS-0017 Exosomal Del 1 as a potent diagnostic marker for breast cancer : A prospective cohort study Soo Jung Lee 1, Jeeyeon Lee 2, Jin Hyang Jung 2, Ho Yong Park 2, Chan Hyeong Lee 3, Pyong Gon Moon 3, Moon Chang Baek 3, Yee Soo Chae 1 1 Department of Oncology/Hematology, 2 Department of Surgery, 3 Department of Molecular Medicine, Cell and Matrix Research Institute, Kyungpook National University, Daegu, Republic of Korea
Background Breast cancer(bc) is the leading cause of death worldwide and is the most common type of cancer in women. As breast screening rates have increased, more cases have been found at earlier stages. Identifying a biomarker to detect BC at an early stage will improve clinical outcomes. After treatment with curative surgery, Identification of patients with residual disease who are at high risk of relapse may be used to stratify patients to adjuvant therapy.
Exosome Extracellular Vesicles such as exosomes are informative membranous vesicles that originate in large multivesicular bodies Interestingly, cancer cells seem to release more EVs than normal cells. It has been shown that plasma samples of patients with advanced lung cancer, ovarian cancer, and colon cancer had higher levels of EVs. Gene Chromosome Cancer, 51 (2012), pp. 409 418; Clin. Lung Cancer, 10 (2009), pp. 42 46; Gynecol. Oncol., 110 (2008), pp. 13 21
DEL 1 : Developmental endothelial locus 1 protein Also known as Epidermal growth factor like repeats and discoidin I like domains 3 (EDIL3) First identified as an extracellular matrix protein expressed by endothelial cells during embryonic vascular development Also shown to promote adhesion of endothelial cells through its interaction with integrin receptors. Integrins can control cellular behavior through FAK and c Src. Recent studies have shown that the FAK Src complex is activated in many tumor cells and lead to tumor growth and metastasis 1. Genes Dev., 12 (1998), pp. 21 33 2. FASEB J., 26 (2012), pp. 3412 3420
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This assay is simple, reproducible, quantitative, and minimally invasive using a small amount of plasma
Optimun Cut off level of Del 1=0.5 ROC curves for Del 1 and CA 15 3 for patients with breast cancer(bc) versus benign breast tumors (bb) and healthy controls(hc) AUC, 0.954; 95% confidence interval (CI), 0.923 0.975; sensitivity at 95% specificity, 73.1% Moon P G et al. Clin Cancer Res; 22(7) April 1, 2016
Del 1 as an early diagnostic biomarker CA 15 3 DEL 1 Moon P G et al. Clin Cancer Res; 22(7) April 1, 2016
Objective Confirming diagnostic role of exosomal Del 1 in a prospective cohort with breast cancer by comparing plasma exosomal Del 1 levels before and after curative surgery. Identifying optimal sampling time Confirmation the role of exosomal Del 1 in a large cohort
Patients Cohort 1 : For optimal sampling time 22 patients with IDC (19) + DCIS (3) Cohort 2 : For confirmation of role of Exosomal Del 1 111 patients with BC prospectively enrolled between May 2013 and June 2015 at Kyungpook National University Medical Center (Daegu, Korea) Underwent R0 resection as curative intent
Method (1) Sample Collection for the optimal time of blood draw To identify the optimal time of sampling after surgery, blood samples were serially collected at post operative day (POD) 1, 3, 5, and 7 (N=22) Preop POD1 POD3 POD5 POD7 0
Method (2) After then, blood sample were serially collected after surgery (N=111) Compare Exosomal Del 1 at pre and post operation Preop POD* 6mo 12 months 0 18 months
Method: ELISA For quantification of Del 1 protein on exosoms in plasma using an ELISA, Circulating exosomes isolated from plasma, using primary capture antibodies (anti CD 63 antibody) and anti Del 1 (ab151308; Abcam) antibody Anti Del 1 Ab Del 1
Results: Patients Characteristics (cohort 1) Characteristics N=22 % Age (median, range) 56 (40-79) Histology IDC 19 86.4 DCIS 3 13.6 Primary tumor site Left 13 59.1 Right 9 40.9 Stage (ps) 0.0 0/1A/1B 3/11/0 13.6/50.0/0 2A/2B 4/2 18.2/9.1 3A 2 9.1 Surgery Mastectomy 5 22.7 Breast conserving 17 77.3 Del-1 at diagnosis 0.987±0.072 > 0.5 21 95.5 0.4-0.5 1 4.5
Results: Serial change of exosomal Del 1 level after curative surgery (N=22) All exosomal Del 1 levels were normalized at POD 1
Results: Patients Characteristics (cohort 2) Characteristics N % Characteristics N % Age (median, range) 50.0, 30-79 Tumor type 40 20 18 Hormone-responsive 65 58.6 41-55 57 51.4 HER2-overexpressing 26 23.4 CA15-3, elevated 1 0.9 Triple negative 20 18.0 Stage (pstage) Surgery 1A/1B 36/2 32.4/1.8 Mastectomy 43 38.7 2A/2B 41/17 36.9/15.3 Breast conserving 68 61.3 3A/3C 9/6 8.1/5.4 Adjuvant chemotherapy 97 87.3 Histological grade CMF 4 3.6 1 9 8.1 Anthracycline 23 20.7 2 63 56.8 taxane 32 28.8 3 39 35.1 Anthracycline + taxane 38 34.2 ER expression Adjuvant trastuzumab 32 28.8 Positive 78.70.3 Adjuvant hormone 63 56.8 PR expression tamoxifen 43 38.7 Positive 70 63.0 Aromatase inhibitors 20 18.0 HER2 Relapse 13 11.7 Positive 26 23.4 Locoregional 6 5.4 Ki67 26.05 ± 27.38 Systemic 7 6.3 14% 57 51.4 Death 2 1.8
Del 1 Characteristics N % Del 1 at diagnosis, mean ± SD 1.23 ± 0.46 > 0.5 107 96.4 0.4 0.5 3 2.7 < 0.4 1 0.9 Del 1, postoperative, mean ± SD 0.19 ± 0.12 > 0.5 6 5.4 0.4 0.5 4 3.6 < 0.4 101 91 Δ (preoperative postoperative) mean ± SD 1.04 ± 0.45
Result Difference of exosomal Del-1 level between baseline and after-surgery
RFS according to the postoperative Del 1 A B C HR 24.0, 95% CI 3.5 163.9, P = 0.0011 HR 32.8, 95% CI 6.4 168.9, P = 0.00003 Cut off value of 0.5 Cut off value of 0.4 Lower del 1 level in plasma after surgery was associated with a better survival. More significant differences with cut of value 0.4. Postop del 1 in plasma may reflect residual tumor burden.
RFS according to the postoperative Del 1 LN BC LN + BC A B P < 0.00001 P = 0.05254 Prognostic role of postop del 1 was identified for the LN negative breast cancer patients
RFS according to the postoperative Del 1 Hormone responsive HER2 overexpressing Triple negative A B C P = 0.00008 P = 0.00064 P = 0.00002
Conclusion As the current prospective cohort study demonstrated a normalization of exosomal Del 1 after curative surgery, exosomal Del 1 can be confirmed as a potent diagnostic biomarker for breast cancer. High Del 1 level after surgery seems related with early relapse suggesting a potential prognostic marker by identifying the existence of residual tumor. The minimally invasive method with high sensitivity and specificity based on the molecular biomarker, Del 1 positive circulating exosomes, is promising for the early diagnosis of breast cancer using only one drop of blood.
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Relapse according to Del 1 level 5.9 % 75.0 % 66.7 %
Correlation with cancer progression Recently implicated in multiple cancers including hepatocellular carcinoma, colorectal cancer, bladder cancer and pancreatic adenocarcinoma Tumor type Author Journal Results HCC Sun et al. World J. Gastroenterol (2010) Del1 expression predict poor prognosis (activates ERK and TGF b signaling, leading to tumor cell invasion and pro liferation) Bladder ca Beckham et al. J.Urol (2014) Del1 facilitate cancer progression (activates EGFR signaling in bladder cancer cells, thereby inducing tumor cell migration) Pancreatic ca Jiang et al. Oncotarget (2016) Overexpressed EDIL3 predicts poor prognosis Lung ca Lee et al. Biochem Biophys Res Commun (2015) Del 1 overexpression potentiates proliferation and invasion The relationship between the Del 1 level and cancer progression is still controversial
Function of Del 1: Invasion assays Neutralization of EDIL3 protein wi th anti EDIL3 antibody resulted in inhibition of invasion J E LEE et al. Journal of proteomics 131 (2016) 17 28
Invasion through the integrin FAK signaling pathway Effect of anti integrin antibodies on MCF7 invasion Del 1 on exosomes promotes breast cancer invasion via integrin FAK signaling Phosphorylated FAK in MCF 7 cells with recombinant EDIL3 FAK: focal adhesion kinase J E LEE et al. Journal of proteomics 131 (2016) 17 28
Del 1 on circulating exosomes