- Email: [email protected]
III breast cancer
Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer Baoquan Liu PhD, Fei Su PhD, Mingwei Chen PhD, Yue Li PhD, Xiuying Qi PhD, Jianbing Xiao PhD, Xuemei Li PhD, Xiangchen Liu MM, Wenlong Liang MM, Yafang Zhang PhD, Jianguo Zhang MD PII: DOI: Reference:
S0046-8177(17)30100-4 doi: 10.1016/j.humpath.2017.03.016 YHUPA 4170
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
6 January 2017 12 March 2017 23 March 2017
Please cite this article as: Liu Baoquan, Su Fei, Chen Mingwei, Li Yue, Qi Xiuying, Xiao Jianbing, Li Xuemei, Liu Xiangchen, Liang Wenlong, Zhang Yafang, Zhang Jianguo, Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer, Human Pathology (2017), doi: 10.1016/j.humpath.2017.03.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer
RI P
Xiangchen Liu3, Wenlong Liang3, Yafang Zhang1, Jianguo Zhang3
T
Baoquan Liu1, Fei Su2, Mingwei Chen1, Yue Li3, Xiuying Qi1, Jianbing Xiao1, Xuemei Li1,
SC
Baoquan Liu, Fei Su and Mingwei Chen are contributed equally to this work
1. Department of Anatomy, Harbin Medical University, Harbin, 150081, P. R. China
MA NU
2. College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, 150081, P. R. China
3. Department of General Surgery, The Second Clinical Hospital, Harbin Medical University,
ED
Harbin, 150081, P. R. China.
Running head: Serum miR-21 and -125b predicting neoadjuvant chemotherapy effect
PT
Correspondence:
Yafang Zhang, Department of Anatomy, Harbin Medical University, 157 Baojian Road, Harbin,
CE
150081, China. (Telephone: +86-0451-86674508. E-mail: [email protected]) Jianguo Zhang, Department of General Surgery, The Second Clinical Hospital, Harbin Medical
AC
University, 246 Xuefu Road, Harbin, 150081, China. (Fax: +86-0451-86605079. E-mail: [email protected]) Author degrees: Baoquan Liu, Fei Su, Mingwei Chen, Yue Li, Xiuying Qi, Jianbing Xiao, Xuemei Li, Yafang Zhang, Doctor of Philosophy (Ph.D); Xiangchen Liu, Wenlong Liang, Master of Medicine (M.M.); Jianguo Zhang, Doctor of Medicine (M.D.) Funding The project sponsored by National Natural Science Foundation of China (Grant number 81372838) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. Conflict of interest The authors declare that they have no conflicts of interest.
ACCEPTED MANUSCRIPT Abstract The predictive value of serum miRNAs (ser-miRNA) for the response to neoadjuvant chemotherapy (NCT) and the prognosis of breast cancer patients was investigated in the current
RI P
T
study. The study included 118 stage II/III breast cancer patients and 30 healthy adult women. Peripheral blood was drawn from participants before the start (baseline, BL), at the end of the second cycle (first evaluation during NCT, FEN), and at the end of NCT (second evaluation during
SC
NCT, SEN). The expression of ser-miRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and their association with chemotherapy
MA NU
response and prognosis was analyzed. MiR-19a, miR-21, miR-125b, miR-155, miR-205, and miR-373 were significantly upregulated in the serum of breast cancer patients at BL, miR-451 was significantly downregulated, and miR-122 were unchanged compared with the levels in healthy women. The expression of ser-miR-125b and the changes of ser-miR-21 expression during NCT
ED
were associated with chemotherapy response and disease-free survival (DFS). In chemotherapy responders, ser-miR-125b expression was lower than that of non-responders at BL, FEN, and SEN,
PT
and ser-miR-21 levels decreased from BL to FEN and from BL to SEN. Survival analysis showed that patients with lower ser-miR-125b expression at BL, FEN, and SEN had favorable DFS, and
CE
those with decreased ser-miR-21 expression from BL to FEN and from BL to SEN had better DFS.
AC
In conclusion, ser-miR-21 and ser-miR-125b were identified as novel, noninvasive predictive markers for NCT response and prognosis in breast cancer. Key words: miR-21; miR-125b; breast cancer; neoadjuvant chemotherapy; prognosis
ACCEPTED MANUSCRIPT Breast cancer is one of the most common malignant cancers among women worldwide and its morbidity is increasing yearly in most countries including China [1]. Surgery, chemotherapy, and endocrine therapy are the major treatments for breast cancer. However, the use of neoadjuvant
T
chemotherapy (NCT) has considerably improved the efficacy of treatment for this disease in
RI P
recent years. Although NCT is an effective therapy for most patients with breast cancer, the benefit is limited in some individuals because of drug resistance [2]. There is currently no reliable
SC
effective method for predicting the chemotherapy response before the start or at the early stage of NCT. Hence, it is an imperative to identify non-invasive and specific biomarkers to assess the
MA NU
effects of NCT at the early stages of therapy. Such biomarkers may be beneficial for the design of personalized treatments and could reduce toxic reactions to chemotherapy. MicroRNAs (miRNAs) are a group of small, non-coding RNAs of 19–22 nucleotides that negatively regulate gene expression post-transcriptionally by specifically binding to the 3′
ED
untranslated region of target mRNAs [3]. miRNAs play important roles in tumorigenesis, metastasis, drug resistance, and the prognosis of certain types of cancer including breast cancer [4].
PT
In recent years, accumulating evidence showed that miRNAs are stably present in the peripheral blood and are non-invasive markers for the diagnosis and prediction of treatment effects in many
CE
types of diseases, especially in cancer [5]. The relationship between serum miRNAs (ser-miRNAs) and treatment response of breast cancer has been reported in several studies, such as miR-19a,
AC
miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373, and miR-451 [6-14]. However, the conclusions from these studies are inconsistent. Therefore, further study in larger patient cohorts is needed to confirm the predictive value of these ser-miRNAs for NCT response. The purpose of the present study was to investigate the value of ser-miRNAs, including miR-19a, miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373, and miR-451 to predict treatment response and prognoses in breast cancer patients receiving NCT.
Materials and Methods Patients and blood samples The present study included 118 diagnosed stage II/III primary breast cancer patients treated between May 2011 and July 2013 at the Department of Breast Surgery of Second Clinical Hospital, Harbin Medical University, Harbin, Heilongjiang, China. The mean age of the patients at the time
ACCEPTED MANUSCRIPT of diagnosis was 45.6 years (range, 32.1-76.6 years). All the patients underwent preoperative NCT with an association of 75 mg/m2 docetaxel, 75 mg/m2 epirubicin, and 500 mg/m2 cytoxan (TEC regimen). All patients received 4~6 cycles of NCT, and each cycle is 21 days. Thirty healthy adult
T
women which were not diagnosed any malignancy previously (as controls) were included in this
RI P
study. Informed consent was obtained from all the objects and the study protocol was approved by the Ethics Committee of Harbin Medical University. Survival data was available for all 118
SC
patients. The median follow-up period after surgery was 25 months (range, 10–36 months). Before the start, at the end of the second cycle, and at the end of NCT, all the patients received a venous
MA NU
blood draw in the antecubital area. Venous blood was placed at room temperature for 2~3 hours, and then centrifuged for 20 minutes at 1,900 g. The upper layer of serum was transferred to a new RNAse-free tube and immediately stored at -80°C until use.
Extraction of total RNA and quantitative reverse transcriptase-polymerase chain reaction
ED
(qRT-PCR)
The miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) was used to isolate total RNA
PT
from 200μL serum according to the manufacturer’s instructions. The extracted total RNA was eluted in 14 μL of RNAse-free water. The concentration and purity of the RNA samples were
CE
measured using the Nanodrop-1000 (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples were immediately stored at -80°C for further use.
AC
Thirty nanograms of total RNA were reverse transcribed into complementary DNA using specific primers for miR-19a, miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373 and miR-451 using the PrimeScript™ RT reagent Kit (Takara Biotechnologies Inc., Tokyo, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed using the SYBR® Premix Ex Taq™ II (Takara Biotechnologies Inc.) on the Stratagene Mx3000P Real-Time qPCR System (Agilent Technologies, Santa Clara, CA, USA). The qRT-PCR reaction was performed as follows: 95°C for 30 s, then 40 cycles of 95°C for 5 s, and 60°C for 30 s. Ser-miR-16 levels were used to normalize the miRNAs expression of interest because of its high stability expression across normal individuals and cancer patients [15]. The relative miRNA expression levels of interest were calculated using the 2 −ΔΔCt method. The ΔCt = the Ct value of miRNA of interest minus the Ct value of miR16, and the ΔΔCt = the Ct value of breast cancer patients minus the Ct
ACCEPTED MANUSCRIPT value of healthy volunteers. Evaluation of NCT efficacy The tumors were estimated by radiologic examination using chest and abdomen
T
computerized tomography (CT)-scans and X-rays. The Response Evaluation Criteria in Solid
RI P
Tumors (RECIST 1.1) was used to evaluate chemotherapeutic responses [16]. The patients having complete response (CR) or partial response (PR) were diveded into the chemotherapy responders,
SC
and the patients having stable disease (SD) or progressive disease (PD) were diveded into the chemotherapy non-responders.
MA NU
Statistical analysis
All data were expressed as the means ± standard deviations and were analyzed with SPSS 18.0 software (SPSS, Chicago, IL, USA). The expression of selected miRNAs between groups was compared using the t-test and one-way analysis of variance. Receiver operating characteristic
ED
(ROC) curves were constructed and the area under the ROC curves (AUC) with 95% confidence intervals (CI) was calculated to evaluate the predictive power of selected miRNAs for the NCT
PT
response. Kaplan–Meier method and Cox proportional hazards model were performed to evaluate the prognostic values of selected miRNAs. P value < 0.05 was considered statistically significant.
CE
Results
Verification of breast cancer and clinicopathological characteristics of the patients
AC
All patients underwent postoperative histological examination for verification of breast cancer (Fig. 1). The clinicopathological characteristics of the patients are summarized in Table 1. The HER2 status of all patients included in this study is negative. Expression of ser-miRNAs in breast cancer patients and healthy controls and their changes during NCT The expression of ser-miRNAs in breast cancer patients before the start of NCT (baseline, BL), at the end of the second cycle (the first evaluation during NCT, FEN), and at the end of NCT (the second evaluation during NCT, SEN) was evaluated by qRT-PCR. The results showed that the levels of ser-miR-19a, ser-miR-21, ser-miR-125b, ser-miR-155, ser-miR-205, and ser-miR-373 were significantly higher in breast cancer patients at BL, ser-miR-451 was significant lower, and ser-miR-122 did not differ from the levels in healthy women (Fig. 2). The relation between
ACCEPTED MANUSCRIPT clinicopathological parameters and ser-miRNAs expression is shown in Table 1. Increased expression of ser-miR-125b and ser-miR-155 was significantly associated with advanced stage and lymph node metastasis. In addition, analysis of changes in the expression of these
T
ser-miRNAs from BL to FEN and from BL to SEN showed no statistically significant differences.
RI P
Ser-miR-21 and ser-miR-125b were associated with the response to NCT
Of 118 patients receiving NCT, 25 patients achieved CR, 55 PR, 31 had SD, and 7 had PD.
SC
Therefore, 80 patients were classified as responders and 38 were non-responders. To examine the relation between ser-miRNA expression and chemotherapy response, the corresponding
MA NU
ser-miRNAs were analyzed in responders and non-responders. The results showed that the serum levels of miR-19a, miR-21, miR-122, miR-155, miR-205, miR-373, and miR-451 at BL, FEN, and SEN did not differ between responders and non-responders. However, a significant association was found between NCT response and ser-miR-125b expression, which was significantly higher at
ED
BL, FEN, and SEN in non-responders than in responders (Fig. 3). Analysis of the relationship between changes in miRNA expression during NCT and treatment response showed that
PT
ser-miR-21 expression changes during NCT were significantly associated with the response to NCT. Although the expression of ser-miR-21 did not change significantly from BL to FEN and
CE
from BL to SEN in all patients, the mean expression levels of ser-miR-21 were significantly lower at FEN and SEN than at BL in responders (P = 0.016 for both) but not in non-responders. In
AC
addition, the changes of ser-miR-21 expression from BL to FEN and from BL to SEN were 0.28 ± 0.24 and 0.27 ± 0.28, respectively, in responders, compared with −0.06 ± 0.11 and −0.04 ± 0.41, respectively, in non-responders (P < 0.001 for both). Of 80 responders, 72 and 70 had decreased expression of ser-miR-21 at FEN and SEN, respectively, than at BL, whereas of 38 non-responders, only 10 and 12 patients had decreased expression of ser-miR-21, and significant differences were found between the two groups (P < 0.001 for FEN and SEN) (Fig. 4). These results indicated that ser-miR-21 and ser-miR125b expression during NCT could predict the treatment response. The predictive power of ser-miR-21 and ser-miR-125b for NCT response To estimate the predictive power of the changes of ser-miR-21 level and ser-miR-125b expression for chemotherapeutic responses, the ROC curves and AUCs were calculated. The AUC
ACCEPTED MANUSCRIPT values for the expression of ser-miR-125b at BL, FEN, and SEN were 0.777, 0.769, and 0.767, respectively, and those for the changes of ser-miRNA-21 from BL to FEN and from BL to SEN were 0.927 and 0.872, respectively. Discriminating responders from non-responders at the early
T
stage of NCT is crucial for improving the treatment effect; therefore, ser-miR-125b expression at
RI P
BL was combined with the change of ser-miR-21 from BL to FEN, and the predictive power of this combination was estimated for chemotherapeutic responders. The AUC for the combination of
SC
the two ser-miRNAs was 0.958 (Fig. 5). Taken together, these results indicated that ser-miR21
non-responders with high accuracy.
MA NU
and ser-miR125b, alone or in combination, can discriminate between responders and
Correlation between ser-miR21 and ser-miR-125b with disease-free survival (DFS) Survival analysis was performed to evaluate the prognostic values of ser-miR-21 and ser-miR-125b. The results showed that changes in ser-miR-21 expression from BL to FEN and
ED
from BL to SEN, and ser-miR-125b expression at BL, FEN, and SEN were significantly associated with DFS. Patients showing decreased ser-miR-21 expression from BL to FEN and
PT
from BL to SEN had better DFS than those with increasing ser-miR-21 expression (P < 0.001 for both), and patients with low ser-miR-125b expression at BL, FEN, and SEN had more favorable
CE
DFS than those with high ser-miR-125b expression (P < 0.001 for BL, FEN, and SEN) (Fig. 6). By contrast, changes in ser-miR-125b expression from BL to FEN and from BL to SEN, and the
AC
expression of ser-miR21 at BL, FEN, and SEN were not associated with DFS. Univariate and multivariate Cox proportional hazards regression analyses were performed to determine the influence of these two ser-miRNAs and clinicopathological characteristics on DFS. Univariate analyses showed that eight variables (TNM stage, lymph node metastasis, chemotherapy response status, change of ser-miR-21 from BL to FEN, change of ser-miR-21 from BL to SEN, and expression of ser-miR-125b at BL, at FEN, and at SEN) were significantly associated with DFS. Multivariate analyses showed that changes of ser-miR-21 from BL to FEN and the expression of ser-miR125b at BL were independent prognostic indicators for DFS (Table 2).
Discussion Accumulating evidence indicates that circulating miRNAs play pivotal roles in the diagnosis, prognosis, and treatment of certain types of cancer including breast cancer [5, 17]. However, there
ACCEPTED MANUSCRIPT is considerable discrepancy regarding the roles of circulating miRNAs across these studies [15,18]. The miRNAs examined in the present study were previously reported to be related to the diagnosis or treatment in breast cancer [6-14]. Our results showed that miR-19a, miR-21, miR-125b,
T
miR-155, miR-205, miR-373, and miR-451 were differentially expressed in patients and healthy
RI P
women, and miR-125b and miR-155 were related to advanced stage and lymph node metastasis. These results suggest that these miRNAs can serve as biomarkers for the detection of breast cancer
SC
and for predicting disease progression. The present study identified two miRNAs that were significantly associated with NCT response, miR-21 and miR-125b. This differs partially from
MA NU
previous research results, which may be due to differences in the subjects and chemotherapy regimens.
Several studies showed that miR-21 expression is higher in breast cancer tissues than in those from healthy women and significantly associated with drug resistance and prognosis. Lee showed
ED
that miR-21 expression is increased in invasive breast ductal carcinomas and related to poor survival [19]. A recent meta-analysis described the relationship of miR-21 with the diagnosis and
PT
prognosis of breast cancer [20]. Mattos-Arruda demonstrated that miR-21 can affect the response to chemotherapy plus trastuzumab by triggering an IL-6/STAT3/NF-κB mediated signaling loop
CE
and activating the PI3K pathway [21]. Bourguignon showed that increased miR-21 production contributed to the upregulation of multidrug-resistance proteins and resulted in chemotherapy
AC
resistance in MCF-7 breast cancer cells [22]. The relationship between miR-21 and chemotherapy resistance was also reported in non-small cell lung [23] and gastric cancers [24]. Despite the number of studies showing that the serum levels of miR-21 are increased in breast cancer and other types of cancer and associated with poor prognosis [25,26], the relationship between ser-miR-21 expression and treatment response remains controversial [7-10]. Müller showed the serum levels of miR-21 are significantly higher in patients with HER2 positive breast cancer before and after chemotherapy than in healthy controls, and that the levels increase further after chemotherapy [8]. However Yadav showed that the serum levels of miR-21 are lower after chemotherapy than before chemotherapy [9]. Yoruker did not detect significant differences in ser-miR-21 levels between pre- and post-chemotherapy breast cancer patients [10]. These studies indicated that circulating miR-21 is not associated with chemotherapy response. Meanwhile, the
ACCEPTED MANUSCRIPT role of miR-125b in breast cancer remains controversial. Some studies showed that miR-125b acts as a tumor suppressor in breast cancer, and its expression is decreased in breast cancer tissues and cells and associated with drug resistance [27,28]. However, other studies showed opposite results
T
[29,30]. For example, Wang found that miR-125b expression is upregulated in chemoresistant
RI P
breast cancer patients compared with that in chemotherapy responsive patients, and this may be due to a high percentage of stem cell-like side population cells in chemoresistant breast cancer
SC
tissues [31]. Circulating miR-125b is associated with treatment effect in some cancers. Wang showed that in breast cancer patients receiving pre-operative NCT, ser-miR-125b expression at the
MA NU
start of NCT was higher in non-responders than in responders [11]. This result was in accordance with that reported by D'Angelo in rectal adenocarcinoma [32]. Our present results showed that serum levels of miR-19a, miR-122, miR-155, miR-205, miR-373, and miR-451 were not related to NCT responses. However, the expression of
ED
ser-miR-125b at BL, FEN, and SEN and changes of ser-miR-21 expression from BL to FEN and from BL to SEN during NCT were significantly related to chemotherapy responses. The results
PT
indicated that patients with low serum levels of miR-125b had better treatment responses than those with high levels, and that patients with decreased levels of ser-miR-21 during NCT had more
CE
favorable treatment responses than those with increased levels. The survival analysis results showed that patients with low ser-miR125b levels and decreased ser-miR-21 during NCT had
AC
better DFS than those with high ser-miR125b and increased ser-miR-21 levels. The ROC curves showed that the these two ser-miRNAs had good discrimination power between responders and non-responders, and combined expression of ser-miR-125b at BL and changes in ser-miR-21 expression from BL to FEN had a better discrimination power and higher accuracy. This indicated that the combination of these two ser-miRNAs could predict the treatment effects at the early stages of NCT. In addition, the present results showed that the changes of ser-miR-21 levels reflected a dynamic process in breast cancer, and therefore could be used to monitor disease recurrence after NCT and surgery. A recent study showed that changes in circulating miRNA expression are associated with treatment response. Hansen [33] demonstrated that decreased ser-miR-126 levels are significantly related to good chemotherapeutic responses and survival in metastatic colorectal cancer patients receiving chemotherapy plus bevacizumab. The mechanism
ACCEPTED MANUSCRIPT underlying this effect could be associated with the closed relationship between miR-126 and vascular endothelial cells, which are specific targets of bevacizumab. However, the mechanism involved in our study remains unclear, and further investigation is necessary.
T
The present study had several limitations. All the patients included in the study were from
RI P
China. Since the study did not include an analysis of these two miRNAs in different ethnic populations, their use as biomarkers in different populations is limited. In addition, the biological
SC
behaviors of breast cancer are largely affected by molecular subtypes [34], and the relationship between these subtypes and miRNAs has been reported in recent years [35]. Therefore, the
MA NU
conflicting conclusions reported in previous studies may in part be attributed to differences in the subtypes of the patients analyzed. In further studies, the analysis should be performed according to molecular subtypes. Finally, the TEC chemotherapy regimen was used in our study, and further verification of our results in different chemotherapy regimens is necessary.
ED
In conclusion, the present study identified novel non-invasive markers for breast cancer. Dynamic monitoring and combination use of ser-miR-125b and ser-miR-21 can predict the
PT
response to NCT and DFS. Our results may be beneficial for the individualization of treatment and
CE
more effective administration of NCT in breast cancer.
Ethical approval All procedures performed in studies involving human participants were in
AC
accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
ACCEPTED MANUSCRIPT References [1] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin 2016; 66: 115-32.
RI P
T
[2] Casey MC, Sweeney KJ, Brown JA, Kerin MJ. Exploring circulating micro-RNA in the neoadjuvant treatment of breast cancer. Int J Cancer 2016; 139: 12-22.
[3] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:
SC
281-97.
[4] Kutanzi KR, Yurchenko OV, Beland FA, Checkhun VF, Pogribny IP. MicroRNA-mediated
MA NU
drug resistance in breast cancer. Clin Epigenetics 2011; 2(2): 171-185. [5] Taslim C, Weng DY, Brasky TM, Dumitrescu RG, Huang K, Kallakury BV, Krishnan S, Llanos AA, Marian C, McElroy J, Schneider SS, Spear SL, Troester MA, Freudenheim JL, Geyer S, Shields PG. Discovery and replication of microRNAs for breast cancer risk using
ED
genome-wide profiling. Oncotarget 2016; 7(52): 86457-86468. [6] Li Q, Liu M, Ma F, Luo Y, Cai R, Wang L, Xu N, Xu B. Circulating miR-19a and miR-205
PT
in serum may predict the sensitivity of luminal A subtype of breast cancerpatients to neoadjuvant chemotherapy with epirubicin plus paclitaxel. PLoS One 2014; 9(8): e104870.
CE
[7] Al-Khanbashi M, Caramuta S, Alajmi AM, Al-Haddabi I, Al-Riyami M, Lui WO, Al-Moundhri
AC
MS Tissue and Serum miRNA Profile in Locally Advanced Breast Cancer (LABC) in Response to Neo-Adjuvant Chemotherapy (NAC) Treatment. PLoS One 2016; 11(4): e0152032.
[8] Müller V, Gade S, Steinbach B, Loibl S, von Minckwitz G, Untch M, Schwedler K, Lübbe K, Schem C, Fasching PA, Mau C, Pantel K, Schwarzenbach H. Changes in serum levels of miR-21, miR-210, and miR-373 in HER2-positive breast cancer patients undergoing neoadjuvant therapy: a translational research project within the Geparquinto trial. Breast Cancer Res Treat 2014; 147(1): 61-8. [9] Yadav P, Mirza M, Nandi K, Jain SK, Kaza RC, Khurana N, Ray PC, Saxena A. Serum microRNA-21 expression as a prognostic and therapeutic biomarker for breast cancer patients. Tumour Biol 2016; 37(11):15275-15282. [10] Yoruker EE, Aydoğan F, Gezer U, Saip P, Dalay N. Analysis of circulating microRNAs during
ACCEPTED MANUSCRIPT adjuvant chemotherapy in patients with luminal A breast cancer. Mol Clin Oncol 2015; 3(4): 954-958. [11] Wu X, Somlo G, Yu Y, Palomares MR, Li AX, Zhou W, Chow A, Yen Y, Rossi JJ, Gao H,
T
Wang J, Yuan YC, Frankel P, Li S, Ashing-Giwa KT, Sun G, Wang Y,Smith R, Robinson K,
RI P
Ren X, Wang SE. De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locallyadvanced breast cancer. J Transl Med 2012; 10: 42.
SC
[12] Wang H, Tan G, Dong L, Cheng L, Li K, Wang Z, Luo H. Circulating miR-125b as a marker predicting chemoresistance in breast cancer. PLoS One 2012; 7(4): e34210.
MA NU
[13] Sun Y, Wang M, Lin G, Sun S, Li X, Qi J, Li J. Serum microRNA-155 as a potential biomarker to track disease in breast cancer. PLoS One 2012; 7(10): e47003. [14] Gu X, Xue JQ, Han SJ, Qian SY, Zhang WH. Circulating microRNA-451 as a predictor of resistance to neoadjuvant chemotherapy in breast cancer. Cancer Biomark 2016; 16(3):
ED
395-403.
[15] Roth C, Rack B, Muller V, Janni W, Pantel K, Schwarzenbach H. Circulating microRNAs as
PT
blood-based markers for patients with primary and metastatic breast cancer. Breast Cancer Res 2010; 12(6): R90.
CE
[16] Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG. New guidelines to evaluate the
AC
response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92(3): 205-16. [17] Huo D, Clayton WM, Yoshimatsu TF, Chen J, Olopade OI. Identification of a circulating MicroRNA signature to distinguish recurrence in breast cancer patients. Oncotarget 2016; 7(34): 55231-55248. [18] Hagrass HA, Sharaf S, Pasha HF, Tantawy EA, Mohamed RH, Kassem R. Circulating microRNAs - a new horizon in molecular diagnosis of breast cancer. Genes Cancer 2015; 6(5-6): 281-7. [19] Lee JA, Lee HY, Lee ES, Kim I, Bae JW. Prognostic Implications of MicroRNA-21 Overexpression in Invasive Ductal Carcinomas of the Breast. J Breast Cancer 2011; 14(4):
ACCEPTED MANUSCRIPT 269-75. [20] Li S, Yang X, Yang J, Zhen J, Zhang D. Serum microRNA-21 as a potential diagnostic biomarker for breast cancer: a systematic review and meta-analysis. Clin Exp Med 2016;
T
16(1): 29-35.
A, Pérez-Garcia
J, Masci
G, Corsi
RI P
[21] De Mattos-Arruda L, Bottai G, Nuciforo PG, Di Tommaso L, Giovannetti E, Peg V, Losurdo F, Cortés
J,Seoane
J, Calin
GA, Santarpia
L.
SC
MicroRNA-21 links epithelial-to-mesenchymal transition and inflammatory signals to confer resistance to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast
MA NU
cancer patients. Oncotarget 2015; 6(35): 37269-80.
[22] Bourguignon LY, Spevak CC, Wong G, Xia W, Gilad E. Hyaluronan-CD44 interaction with protein kinase C (epsilon) promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21, leading to down-regulation of the tumor suppressor protein
284(39): 26533-46.
ED
PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells. J Biol Chem 2009;
PT
[23] Gao W, Lu X, Liu L, Xu J, Feng D, Shu Y. MiRNA-21: a biomarker predictive for platinum-based adjuvant chemotherapy response in patients with non-small cell lung cancer.
CE
Cancer Biol Ther 2012; 13(5): 330-40. [24] Jin B, Liu Y, Wang H. Antagonism of miRNA-21 Sensitizes Human Gastric Cancer Cells to
AC
Paclitaxel. Cell Biochem Biophys 2015; 72: 275-82. [25] Wang B, Zhang Q. The expression and clinical significance of circulating microRNA-21 in serum of five solid tumors. J Cancer Res Clin Oncol 2012; 138: 1659-66. [26] Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DS. Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin Chem 2011; 57: 84-91. [27] Xie X, Hu Y, Xu L, Fu Y, Tu J, Zhao H, Zhang S, Hong R, Gu X. The role of miR-125b -mitochondria-caspase-3 pathway in doxorubicin resistance and therapy in human breast cancer. Tumour Biol 2015; 36: 7185-94. [28] Yang Q, Wang Y, Lu X, Zhao Z, Zhu L, Chen S, Wu Q, Chen C, Wang Z. MiR-125b regulates epithelial-mesenchymal transition via targeting Sema4C in paclitaxel-resistant breast
ACCEPTED MANUSCRIPT cancer cells. Oncotarget 2015; 6: 3268-79. [29] Vilquin P, Donini CF, Villedieu M, Grisard E, Corbo L, Bachelot T, Vendrell JA, Cohen PA.
poor prognosis in breast cancer. Breast Cancer Res 2015; 17: 13.
T
MicroRNA-125b upregulation confers aromatase inhibitor resistance and is a novel marker of
RI P
[30] Tang F, Zhang R, He Y, Zou M, Guo L, Xi T. MicroRNA-125b induces metastasis by targeting STARD13 in MCF-7 and MDA-MB-231 breast cancer cells. PLoS ONE 2012; 7:
SC
e35435.
[31] Wang HJ, Guo YQ, Tan G, Dong L, Cheng L, Li KJ, Wang ZY, Luo HF. miR-125b regulates
Biochem 2013; 114: 2248-57.
MA NU
side population in breast cancer and confers a chemoresistant phenotype. J Cell
[32] D'Angelo E, Fassan M, Maretto I, Pucciarelli S, Zanon C, Digito M, Rugge M, Nitti D, Agostini M. Serum miR-125b is a non-invasive predictive biomarker of the pre-operative
ED
chemoradiotherapy responsiveness in patients with rectal adenocarcinoma. Oncotarget 2016; 7: 28647-57.
PT
[33] Hansen TF, Carlsen AL, Heegaard NH, Sørensen FB, Jakobsen A. Changes in circulating microRNA-126 during treatment with chemotherapy and bevacizumab predictstreatment
CE
response in patients with metastatic colorectal cancer. Br J Cancer 2015; 112: 624-629. [34] Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J, Shi B. Breast cancer intrinsic subtype
AC
classification, clinical use and future trends. Am J Cancer Res 2015; 5: 2929-2943. [35] Haakensen VD, Nygaard V, Greger L, Aure MR, Fromm B, Bukholm IR, Lüders T, Chin SF, Git A, Caldas C, Kristensen VN,Brazma A, Børresen-Dale AL, Hovig E, Helland Å. Subtype-specific micro-RNA expression signatures in breast cancer progression. Int J Cancer 2016; 139: 1117-28.
ACCEPTED MANUSCRIPT
PT
Table 1 Clinicopathologic characteristic and baseline serum miRNAs expression levels of breast cancer patients
n miR-19a
miR-205
miR-451
0.70(0.31)
miR-21
miR-155
miR-373
miR-125b
0.94(0.27)
3.30(0.62)
5.41(1.84)
2.71(0.91)
0.51(1.99)
1.00(0.35)
3.28(0.55)
5.49(1.66)
2.75(0.84)
0.51(2.17)
miR-122
NU S
Characteristic
CR I
Mean of serum miRNAs expression（Standard deviation）
Age (years) 78
6.55(2.34)
5.35(1.79)
<40
40
6.70(1.99)
5.61(1.75)
Invasive ductal carcinoma
90
6.62(2.68)
5.31(1.73)
0.70(0.30)
0.96(0.31)
3.26(0.57)
5.4(1.8)
2.68(0.91)
4.97(1.92)
Others
28
6.53(2.11)
5.84(1.88)
0.66(0.21)
0.98(0.26)
3.37(0.67)
5.5(1.7)
2.86(0.81)
5.50(2.37)
Well
37
6.79(2.21)
5.23(1.48)
0.68(0.21)
1.04(0.22)
3.25(0.64)
5.55(1.49)
2.77(0.75)
5.17(2.05)
Moderate
54
6.66(2.42)
5.60(1.96)
0.71(0.32)
0.90(0.30)
3.35(0.57)
5.55(2.02)
2.70(0.95)
5.00(1.99)
MA
≥40
D
0.69(0.24)
AC
Differentiation
CE P
TE
Histological type
ACCEPTED MANUSCRIPT
6.23(1.82)
5.40(1.80)
0.68(0.31)
Ⅱ
62
6.50(2.28)
5.45(1.92)
0.70(0.32)
Ⅲ
56
6.71(2.17)
5.43(1.61)
0.69(0.24)
Positive
59
6.74(2.14)
5.35(1.59)
Negative
59
6.46(2.31)
5.53(1.95)
Positive
89
6.58(2.19)
Negative
29
Positive Negative
0.98(0.37)
3.22(0.60)
5.07(1.61)
2.68(0.97)
5.18(2.20)
3.32(0.59)
4.12(1.10) a
2.59(0.82)
3.82(1.23) a
0.96(0.28)
3.25(0.61)
6.89(1.13) a
2.87(0.94)
6.50(1.82) a
0.95(0.28)
3.25(0.60)
6.85(1.15) a
0.29(0.93)
6.42(1.84) a
0.70(0.33)
0.97(0.32)
3.33(0.60)
4.03(1.00) a
0.26(0.82)
3.76(1.19) a
5.32(1.69)a
0.67(0.24)
0.92(0.28)
3.32(0.60)
5.62(1.74)
2.69(0.89)
5.12(2.00)
6.66(2.37)
8.81(2.00)a
0.76(0.39)
1.08(0.36)
3.19(0.60)
4.88(1.78)
2.81(0.88)
5.00(2.19)
78
6.57(2.10)
5.38(1.72)
0.70(0.27)
0.92(0.27)
3.31(0.56)
5.28(1.70)
2.62(0.87)
4.89(2.06)
40
6.65(2.48)
5.56(1.89)
0.67(0.30)
1.05(0.33)
3.23(0.68)
5.75(1.89)
2.92(0.90)
5.48(1.97)
PT
27
MA
0.68(0.23)
CE P
AC
ER status
TE
D
Lymph node matastasis
0.97(0.32)
NU S
Stage
CR I
Poor
PR status
ACCEPTED MANUSCRIPT
—
—
—
—
—
—
—
—
Negative
118
6.60(2.22)
5.44(1.77)
0.69(0.28)
0.96(0.30)
3.29(0.60)
5.44(1.77)
2.72(0.89)
5.09(2.04)
Premenopause
55
6.39(1.94)
5.54(1.70)
0.68(0.23)
1.01(0.32)
3.22(0.50)
5.52(1.66)
2.70(0.84)
5.17(2.21)
Postmenopause
63
6.79(2.44)
5.35(1.85)
0.71(0.33)
0.92(0.27)
3.34(0.67)
5.37(1.88)
2.74(0.93)
5.02(1.90)
CR+PR
80
6.49(2.13)
5.33(1.66)
0.70(0.30)
0.93(0.26)
3.33(0.63)
5.33(1.66)
2.67(0.83)
4.42(1.49)a
SD+PD
38
6.84(2.42)
5.67(2.01)
0.68(0.25)
1.02(0.37)
3.21(0.52)
5.67(2.01)
2.83(1.00)
6.52(2.31)a
CR I
0
NU S
Positive
PT
HER2 stutus
TE
D
MA
Menopausal status
AC
CE P
Chemotherapy response status
ER, estrogen receptor; PR, progestrone receptor; HER2, human epidermal growth factor receptor 2. a
P < 0.01.
ACCEPTED MANUSCRIPT Table 2 Cox proportional hazards models analysis for DFS in the patients with primary breast cancer Multivariate analysis
HR
95% CI
P
Age
1.085
0.624~1.887
0.773
Histological type
0.766
0.418~1.403
SC
RI P
T
Univariate analysis
Differentiation
0.982
TNM stage
3.471
Lymph node metastasis
3.341
ER expression
1.100
95% CI
P
MA NU
0.388
0.687~1.403
0.921
1.986~6.066
<0.001
2.382
0.347~16.347
0.377
1.898~5.881
<0.001
0.912
0.107~7.747
0.933
0.592~2.046
0.763
ED 0.624
0.366~1.064
0.083
1.032
0.611~1.744
0.905
0.193
0.112~0.332
<0.001
1.291
0.610~2.731
0.504
8.390
4.500~15.644
<0.001
124.393
10.431~1483.399
<0.001
Ser-miR-125b expression at FEN
7.783
4.235~14.303
<0.001
0.209
0.015~2.833
0.239
Ser-miR-125b expression at SEN
6.598
3.673~11.850
<0.001
0.846
0.133~5.398
0.859
8.751
4.977~15.388
<0.001
51.579
13.942~190.820
<0.001
7.386
4.260~12.805
<0.001
1.696
0.794~3.623
0.173
Menopause status
CE
Chemotherapy response
PT
PR expression
HR
AC
Ser-miR-125b expression at BL
Change of Ser-miR-21 from BL to FEN Change of Ser-miR-21 from BL to SEN
ACCEPTED MANUSCRIPT Fig. 1 HE staining in breast cancer tissues. A. Invasive ductal carcinoma, ×200. B. Invasive lobular carcinoma, ×200.
RI P
T
Fig. 2 Serum levels of miRNAs in breast cancer patients and healthy controls. **P < 0.01 compared to healthy controls; #P < 0.05 compared to clinical stageⅡ.
SC
Fig. 3 Serum levels of miR-125b in responing and non-responding patients during the NCT. A. Serum levels of miR-125b in responing and non-responding patients at BL, FEN and SEN. B.
MA NU
Changes in ser-miR-125b levels from BL to FEN and from BL to SEN in responing and non-responding patients, **P < 0.01 compared to non-responding patients. Fig. 4 Serum levels of miR-21 in responing and non-responding patients during the NCT. A.
ED
Serum levels of miR-21 in responing and non-responding patients at BL, FEN and SEN. B.
PT
Changes in serum miR-21 levels from BL to FEN and from BL to SEN in responing and non-responding patients. C, D, E, F. Changes in serum miR-21 levels from BL to FEN and from
CE
BL to SEN in every patient. Black lines: decreased ser-miR-21 level; red lines: increased
AC
ser-miR-21 level. *P < 0.05 compared to BL. Fig. 5 ROC curve analysis based on serum levels of miR-125b and changes of miR-21 serum levels for the discrimination between responding and non-responding patients. A. Serum levels of miR-125b at BL. B. Serum levels of miR-125b at FEN. C. Serum levels of miR-125b at SEN. D. Changes of miR-21 serum levels from BL to FEN. E. Changes of miR-21 serum levels from BL to SEN. F. Combination miR-125b levels at BL with the changes of miR-21 levels from BL to FEN. Fig.6 Kaplan-Meier survival analysis for DFS depending on serum levels of miR-125b and changes of miR-21 serum levels. A. Serum levels of miR-125b at BL. B. Serum level of miR-125b at FEN. C. Serum level of miR-125b at SEN. D. Changes of miR-21 serum levels from BL to
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
FEN. E. Changes of miR-21 serum levels from BL to SEN.
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
Figure 1
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 2
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 3
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 4
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 5
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 6
ACCEPTED MANUSCRIPT Highlights 1. MiR-125b and miR-155 are upregulated in the serum of breast cancer patients.
T
2. Ser-iR-21 and miR-125b are associated with chemotherapy response and DFS.
AC
CE
PT
ED
MA NU
SC
RI P
3. MiR-21 and -125b are noninvasive predictive markers for NCT response and prognosis.
S0046-8177(17)30100-4 doi: 10.1016/j.humpath.2017.03.016 YHUPA 4170
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
6 January 2017 12 March 2017 23 March 2017
Please cite this article as: Liu Baoquan, Su Fei, Chen Mingwei, Li Yue, Qi Xiuying, Xiao Jianbing, Li Xuemei, Liu Xiangchen, Liang Wenlong, Zhang Yafang, Zhang Jianguo, Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer, Human Pathology (2017), doi: 10.1016/j.humpath.2017.03.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer
RI P
Xiangchen Liu3, Wenlong Liang3, Yafang Zhang1, Jianguo Zhang3
T
Baoquan Liu1, Fei Su2, Mingwei Chen1, Yue Li3, Xiuying Qi1, Jianbing Xiao1, Xuemei Li1,
SC
Baoquan Liu, Fei Su and Mingwei Chen are contributed equally to this work
1. Department of Anatomy, Harbin Medical University, Harbin, 150081, P. R. China
MA NU
2. College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, 150081, P. R. China
3. Department of General Surgery, The Second Clinical Hospital, Harbin Medical University,
ED
Harbin, 150081, P. R. China.
Running head: Serum miR-21 and -125b predicting neoadjuvant chemotherapy effect
PT
Correspondence:
Yafang Zhang, Department of Anatomy, Harbin Medical University, 157 Baojian Road, Harbin,
CE
150081, China. (Telephone: +86-0451-86674508. E-mail: [email protected]) Jianguo Zhang, Department of General Surgery, The Second Clinical Hospital, Harbin Medical
AC
University, 246 Xuefu Road, Harbin, 150081, China. (Fax: +86-0451-86605079. E-mail: [email protected]) Author degrees: Baoquan Liu, Fei Su, Mingwei Chen, Yue Li, Xiuying Qi, Jianbing Xiao, Xuemei Li, Yafang Zhang, Doctor of Philosophy (Ph.D); Xiangchen Liu, Wenlong Liang, Master of Medicine (M.M.); Jianguo Zhang, Doctor of Medicine (M.D.) Funding The project sponsored by National Natural Science Foundation of China (Grant number 81372838) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. Conflict of interest The authors declare that they have no conflicts of interest.
ACCEPTED MANUSCRIPT Abstract The predictive value of serum miRNAs (ser-miRNA) for the response to neoadjuvant chemotherapy (NCT) and the prognosis of breast cancer patients was investigated in the current
RI P
T
study. The study included 118 stage II/III breast cancer patients and 30 healthy adult women. Peripheral blood was drawn from participants before the start (baseline, BL), at the end of the second cycle (first evaluation during NCT, FEN), and at the end of NCT (second evaluation during
SC
NCT, SEN). The expression of ser-miRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and their association with chemotherapy
MA NU
response and prognosis was analyzed. MiR-19a, miR-21, miR-125b, miR-155, miR-205, and miR-373 were significantly upregulated in the serum of breast cancer patients at BL, miR-451 was significantly downregulated, and miR-122 were unchanged compared with the levels in healthy women. The expression of ser-miR-125b and the changes of ser-miR-21 expression during NCT
ED
were associated with chemotherapy response and disease-free survival (DFS). In chemotherapy responders, ser-miR-125b expression was lower than that of non-responders at BL, FEN, and SEN,
PT
and ser-miR-21 levels decreased from BL to FEN and from BL to SEN. Survival analysis showed that patients with lower ser-miR-125b expression at BL, FEN, and SEN had favorable DFS, and
CE
those with decreased ser-miR-21 expression from BL to FEN and from BL to SEN had better DFS.
AC
In conclusion, ser-miR-21 and ser-miR-125b were identified as novel, noninvasive predictive markers for NCT response and prognosis in breast cancer. Key words: miR-21; miR-125b; breast cancer; neoadjuvant chemotherapy; prognosis
ACCEPTED MANUSCRIPT Breast cancer is one of the most common malignant cancers among women worldwide and its morbidity is increasing yearly in most countries including China [1]. Surgery, chemotherapy, and endocrine therapy are the major treatments for breast cancer. However, the use of neoadjuvant
T
chemotherapy (NCT) has considerably improved the efficacy of treatment for this disease in
RI P
recent years. Although NCT is an effective therapy for most patients with breast cancer, the benefit is limited in some individuals because of drug resistance [2]. There is currently no reliable
SC
effective method for predicting the chemotherapy response before the start or at the early stage of NCT. Hence, it is an imperative to identify non-invasive and specific biomarkers to assess the
MA NU
effects of NCT at the early stages of therapy. Such biomarkers may be beneficial for the design of personalized treatments and could reduce toxic reactions to chemotherapy. MicroRNAs (miRNAs) are a group of small, non-coding RNAs of 19–22 nucleotides that negatively regulate gene expression post-transcriptionally by specifically binding to the 3′
ED
untranslated region of target mRNAs [3]. miRNAs play important roles in tumorigenesis, metastasis, drug resistance, and the prognosis of certain types of cancer including breast cancer [4].
PT
In recent years, accumulating evidence showed that miRNAs are stably present in the peripheral blood and are non-invasive markers for the diagnosis and prediction of treatment effects in many
CE
types of diseases, especially in cancer [5]. The relationship between serum miRNAs (ser-miRNAs) and treatment response of breast cancer has been reported in several studies, such as miR-19a,
AC
miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373, and miR-451 [6-14]. However, the conclusions from these studies are inconsistent. Therefore, further study in larger patient cohorts is needed to confirm the predictive value of these ser-miRNAs for NCT response. The purpose of the present study was to investigate the value of ser-miRNAs, including miR-19a, miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373, and miR-451 to predict treatment response and prognoses in breast cancer patients receiving NCT.
Materials and Methods Patients and blood samples The present study included 118 diagnosed stage II/III primary breast cancer patients treated between May 2011 and July 2013 at the Department of Breast Surgery of Second Clinical Hospital, Harbin Medical University, Harbin, Heilongjiang, China. The mean age of the patients at the time
ACCEPTED MANUSCRIPT of diagnosis was 45.6 years (range, 32.1-76.6 years). All the patients underwent preoperative NCT with an association of 75 mg/m2 docetaxel, 75 mg/m2 epirubicin, and 500 mg/m2 cytoxan (TEC regimen). All patients received 4~6 cycles of NCT, and each cycle is 21 days. Thirty healthy adult
T
women which were not diagnosed any malignancy previously (as controls) were included in this
RI P
study. Informed consent was obtained from all the objects and the study protocol was approved by the Ethics Committee of Harbin Medical University. Survival data was available for all 118
SC
patients. The median follow-up period after surgery was 25 months (range, 10–36 months). Before the start, at the end of the second cycle, and at the end of NCT, all the patients received a venous
MA NU
blood draw in the antecubital area. Venous blood was placed at room temperature for 2~3 hours, and then centrifuged for 20 minutes at 1,900 g. The upper layer of serum was transferred to a new RNAse-free tube and immediately stored at -80°C until use.
Extraction of total RNA and quantitative reverse transcriptase-polymerase chain reaction
ED
(qRT-PCR)
The miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) was used to isolate total RNA
PT
from 200μL serum according to the manufacturer’s instructions. The extracted total RNA was eluted in 14 μL of RNAse-free water. The concentration and purity of the RNA samples were
CE
measured using the Nanodrop-1000 (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples were immediately stored at -80°C for further use.
AC
Thirty nanograms of total RNA were reverse transcribed into complementary DNA using specific primers for miR-19a, miR-21, miR-122, miR-125b, miR-155, miR-205, miR-373 and miR-451 using the PrimeScript™ RT reagent Kit (Takara Biotechnologies Inc., Tokyo, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed using the SYBR® Premix Ex Taq™ II (Takara Biotechnologies Inc.) on the Stratagene Mx3000P Real-Time qPCR System (Agilent Technologies, Santa Clara, CA, USA). The qRT-PCR reaction was performed as follows: 95°C for 30 s, then 40 cycles of 95°C for 5 s, and 60°C for 30 s. Ser-miR-16 levels were used to normalize the miRNAs expression of interest because of its high stability expression across normal individuals and cancer patients [15]. The relative miRNA expression levels of interest were calculated using the 2 −ΔΔCt method. The ΔCt = the Ct value of miRNA of interest minus the Ct value of miR16, and the ΔΔCt = the Ct value of breast cancer patients minus the Ct
ACCEPTED MANUSCRIPT value of healthy volunteers. Evaluation of NCT efficacy The tumors were estimated by radiologic examination using chest and abdomen
T
computerized tomography (CT)-scans and X-rays. The Response Evaluation Criteria in Solid
RI P
Tumors (RECIST 1.1) was used to evaluate chemotherapeutic responses [16]. The patients having complete response (CR) or partial response (PR) were diveded into the chemotherapy responders,
SC
and the patients having stable disease (SD) or progressive disease (PD) were diveded into the chemotherapy non-responders.
MA NU
Statistical analysis
All data were expressed as the means ± standard deviations and were analyzed with SPSS 18.0 software (SPSS, Chicago, IL, USA). The expression of selected miRNAs between groups was compared using the t-test and one-way analysis of variance. Receiver operating characteristic
ED
(ROC) curves were constructed and the area under the ROC curves (AUC) with 95% confidence intervals (CI) was calculated to evaluate the predictive power of selected miRNAs for the NCT
PT
response. Kaplan–Meier method and Cox proportional hazards model were performed to evaluate the prognostic values of selected miRNAs. P value < 0.05 was considered statistically significant.
CE
Results
Verification of breast cancer and clinicopathological characteristics of the patients
AC
All patients underwent postoperative histological examination for verification of breast cancer (Fig. 1). The clinicopathological characteristics of the patients are summarized in Table 1. The HER2 status of all patients included in this study is negative. Expression of ser-miRNAs in breast cancer patients and healthy controls and their changes during NCT The expression of ser-miRNAs in breast cancer patients before the start of NCT (baseline, BL), at the end of the second cycle (the first evaluation during NCT, FEN), and at the end of NCT (the second evaluation during NCT, SEN) was evaluated by qRT-PCR. The results showed that the levels of ser-miR-19a, ser-miR-21, ser-miR-125b, ser-miR-155, ser-miR-205, and ser-miR-373 were significantly higher in breast cancer patients at BL, ser-miR-451 was significant lower, and ser-miR-122 did not differ from the levels in healthy women (Fig. 2). The relation between
ACCEPTED MANUSCRIPT clinicopathological parameters and ser-miRNAs expression is shown in Table 1. Increased expression of ser-miR-125b and ser-miR-155 was significantly associated with advanced stage and lymph node metastasis. In addition, analysis of changes in the expression of these
T
ser-miRNAs from BL to FEN and from BL to SEN showed no statistically significant differences.
RI P
Ser-miR-21 and ser-miR-125b were associated with the response to NCT
Of 118 patients receiving NCT, 25 patients achieved CR, 55 PR, 31 had SD, and 7 had PD.
SC
Therefore, 80 patients were classified as responders and 38 were non-responders. To examine the relation between ser-miRNA expression and chemotherapy response, the corresponding
MA NU
ser-miRNAs were analyzed in responders and non-responders. The results showed that the serum levels of miR-19a, miR-21, miR-122, miR-155, miR-205, miR-373, and miR-451 at BL, FEN, and SEN did not differ between responders and non-responders. However, a significant association was found between NCT response and ser-miR-125b expression, which was significantly higher at
ED
BL, FEN, and SEN in non-responders than in responders (Fig. 3). Analysis of the relationship between changes in miRNA expression during NCT and treatment response showed that
PT
ser-miR-21 expression changes during NCT were significantly associated with the response to NCT. Although the expression of ser-miR-21 did not change significantly from BL to FEN and
CE
from BL to SEN in all patients, the mean expression levels of ser-miR-21 were significantly lower at FEN and SEN than at BL in responders (P = 0.016 for both) but not in non-responders. In
AC
addition, the changes of ser-miR-21 expression from BL to FEN and from BL to SEN were 0.28 ± 0.24 and 0.27 ± 0.28, respectively, in responders, compared with −0.06 ± 0.11 and −0.04 ± 0.41, respectively, in non-responders (P < 0.001 for both). Of 80 responders, 72 and 70 had decreased expression of ser-miR-21 at FEN and SEN, respectively, than at BL, whereas of 38 non-responders, only 10 and 12 patients had decreased expression of ser-miR-21, and significant differences were found between the two groups (P < 0.001 for FEN and SEN) (Fig. 4). These results indicated that ser-miR-21 and ser-miR125b expression during NCT could predict the treatment response. The predictive power of ser-miR-21 and ser-miR-125b for NCT response To estimate the predictive power of the changes of ser-miR-21 level and ser-miR-125b expression for chemotherapeutic responses, the ROC curves and AUCs were calculated. The AUC
ACCEPTED MANUSCRIPT values for the expression of ser-miR-125b at BL, FEN, and SEN were 0.777, 0.769, and 0.767, respectively, and those for the changes of ser-miRNA-21 from BL to FEN and from BL to SEN were 0.927 and 0.872, respectively. Discriminating responders from non-responders at the early
T
stage of NCT is crucial for improving the treatment effect; therefore, ser-miR-125b expression at
RI P
BL was combined with the change of ser-miR-21 from BL to FEN, and the predictive power of this combination was estimated for chemotherapeutic responders. The AUC for the combination of
SC
the two ser-miRNAs was 0.958 (Fig. 5). Taken together, these results indicated that ser-miR21
non-responders with high accuracy.
MA NU
and ser-miR125b, alone or in combination, can discriminate between responders and
Correlation between ser-miR21 and ser-miR-125b with disease-free survival (DFS) Survival analysis was performed to evaluate the prognostic values of ser-miR-21 and ser-miR-125b. The results showed that changes in ser-miR-21 expression from BL to FEN and
ED
from BL to SEN, and ser-miR-125b expression at BL, FEN, and SEN were significantly associated with DFS. Patients showing decreased ser-miR-21 expression from BL to FEN and
PT
from BL to SEN had better DFS than those with increasing ser-miR-21 expression (P < 0.001 for both), and patients with low ser-miR-125b expression at BL, FEN, and SEN had more favorable
CE
DFS than those with high ser-miR-125b expression (P < 0.001 for BL, FEN, and SEN) (Fig. 6). By contrast, changes in ser-miR-125b expression from BL to FEN and from BL to SEN, and the
AC
expression of ser-miR21 at BL, FEN, and SEN were not associated with DFS. Univariate and multivariate Cox proportional hazards regression analyses were performed to determine the influence of these two ser-miRNAs and clinicopathological characteristics on DFS. Univariate analyses showed that eight variables (TNM stage, lymph node metastasis, chemotherapy response status, change of ser-miR-21 from BL to FEN, change of ser-miR-21 from BL to SEN, and expression of ser-miR-125b at BL, at FEN, and at SEN) were significantly associated with DFS. Multivariate analyses showed that changes of ser-miR-21 from BL to FEN and the expression of ser-miR125b at BL were independent prognostic indicators for DFS (Table 2).
Discussion Accumulating evidence indicates that circulating miRNAs play pivotal roles in the diagnosis, prognosis, and treatment of certain types of cancer including breast cancer [5, 17]. However, there
ACCEPTED MANUSCRIPT is considerable discrepancy regarding the roles of circulating miRNAs across these studies [15,18]. The miRNAs examined in the present study were previously reported to be related to the diagnosis or treatment in breast cancer [6-14]. Our results showed that miR-19a, miR-21, miR-125b,
T
miR-155, miR-205, miR-373, and miR-451 were differentially expressed in patients and healthy
RI P
women, and miR-125b and miR-155 were related to advanced stage and lymph node metastasis. These results suggest that these miRNAs can serve as biomarkers for the detection of breast cancer
SC
and for predicting disease progression. The present study identified two miRNAs that were significantly associated with NCT response, miR-21 and miR-125b. This differs partially from
MA NU
previous research results, which may be due to differences in the subjects and chemotherapy regimens.
Several studies showed that miR-21 expression is higher in breast cancer tissues than in those from healthy women and significantly associated with drug resistance and prognosis. Lee showed
ED
that miR-21 expression is increased in invasive breast ductal carcinomas and related to poor survival [19]. A recent meta-analysis described the relationship of miR-21 with the diagnosis and
PT
prognosis of breast cancer [20]. Mattos-Arruda demonstrated that miR-21 can affect the response to chemotherapy plus trastuzumab by triggering an IL-6/STAT3/NF-κB mediated signaling loop
CE
and activating the PI3K pathway [21]. Bourguignon showed that increased miR-21 production contributed to the upregulation of multidrug-resistance proteins and resulted in chemotherapy
AC
resistance in MCF-7 breast cancer cells [22]. The relationship between miR-21 and chemotherapy resistance was also reported in non-small cell lung [23] and gastric cancers [24]. Despite the number of studies showing that the serum levels of miR-21 are increased in breast cancer and other types of cancer and associated with poor prognosis [25,26], the relationship between ser-miR-21 expression and treatment response remains controversial [7-10]. Müller showed the serum levels of miR-21 are significantly higher in patients with HER2 positive breast cancer before and after chemotherapy than in healthy controls, and that the levels increase further after chemotherapy [8]. However Yadav showed that the serum levels of miR-21 are lower after chemotherapy than before chemotherapy [9]. Yoruker did not detect significant differences in ser-miR-21 levels between pre- and post-chemotherapy breast cancer patients [10]. These studies indicated that circulating miR-21 is not associated with chemotherapy response. Meanwhile, the
ACCEPTED MANUSCRIPT role of miR-125b in breast cancer remains controversial. Some studies showed that miR-125b acts as a tumor suppressor in breast cancer, and its expression is decreased in breast cancer tissues and cells and associated with drug resistance [27,28]. However, other studies showed opposite results
T
[29,30]. For example, Wang found that miR-125b expression is upregulated in chemoresistant
RI P
breast cancer patients compared with that in chemotherapy responsive patients, and this may be due to a high percentage of stem cell-like side population cells in chemoresistant breast cancer
SC
tissues [31]. Circulating miR-125b is associated with treatment effect in some cancers. Wang showed that in breast cancer patients receiving pre-operative NCT, ser-miR-125b expression at the
MA NU
start of NCT was higher in non-responders than in responders [11]. This result was in accordance with that reported by D'Angelo in rectal adenocarcinoma [32]. Our present results showed that serum levels of miR-19a, miR-122, miR-155, miR-205, miR-373, and miR-451 were not related to NCT responses. However, the expression of
ED
ser-miR-125b at BL, FEN, and SEN and changes of ser-miR-21 expression from BL to FEN and from BL to SEN during NCT were significantly related to chemotherapy responses. The results
PT
indicated that patients with low serum levels of miR-125b had better treatment responses than those with high levels, and that patients with decreased levels of ser-miR-21 during NCT had more
CE
favorable treatment responses than those with increased levels. The survival analysis results showed that patients with low ser-miR125b levels and decreased ser-miR-21 during NCT had
AC
better DFS than those with high ser-miR125b and increased ser-miR-21 levels. The ROC curves showed that the these two ser-miRNAs had good discrimination power between responders and non-responders, and combined expression of ser-miR-125b at BL and changes in ser-miR-21 expression from BL to FEN had a better discrimination power and higher accuracy. This indicated that the combination of these two ser-miRNAs could predict the treatment effects at the early stages of NCT. In addition, the present results showed that the changes of ser-miR-21 levels reflected a dynamic process in breast cancer, and therefore could be used to monitor disease recurrence after NCT and surgery. A recent study showed that changes in circulating miRNA expression are associated with treatment response. Hansen [33] demonstrated that decreased ser-miR-126 levels are significantly related to good chemotherapeutic responses and survival in metastatic colorectal cancer patients receiving chemotherapy plus bevacizumab. The mechanism
ACCEPTED MANUSCRIPT underlying this effect could be associated with the closed relationship between miR-126 and vascular endothelial cells, which are specific targets of bevacizumab. However, the mechanism involved in our study remains unclear, and further investigation is necessary.
T
The present study had several limitations. All the patients included in the study were from
RI P
China. Since the study did not include an analysis of these two miRNAs in different ethnic populations, their use as biomarkers in different populations is limited. In addition, the biological
SC
behaviors of breast cancer are largely affected by molecular subtypes [34], and the relationship between these subtypes and miRNAs has been reported in recent years [35]. Therefore, the
MA NU
conflicting conclusions reported in previous studies may in part be attributed to differences in the subtypes of the patients analyzed. In further studies, the analysis should be performed according to molecular subtypes. Finally, the TEC chemotherapy regimen was used in our study, and further verification of our results in different chemotherapy regimens is necessary.
ED
In conclusion, the present study identified novel non-invasive markers for breast cancer. Dynamic monitoring and combination use of ser-miR-125b and ser-miR-21 can predict the
PT
response to NCT and DFS. Our results may be beneficial for the individualization of treatment and
CE
more effective administration of NCT in breast cancer.
Ethical approval All procedures performed in studies involving human participants were in
AC
accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
ACCEPTED MANUSCRIPT References [1] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin 2016; 66: 115-32.
RI P
T
[2] Casey MC, Sweeney KJ, Brown JA, Kerin MJ. Exploring circulating micro-RNA in the neoadjuvant treatment of breast cancer. Int J Cancer 2016; 139: 12-22.
[3] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:
SC
281-97.
[4] Kutanzi KR, Yurchenko OV, Beland FA, Checkhun VF, Pogribny IP. MicroRNA-mediated
MA NU
drug resistance in breast cancer. Clin Epigenetics 2011; 2(2): 171-185. [5] Taslim C, Weng DY, Brasky TM, Dumitrescu RG, Huang K, Kallakury BV, Krishnan S, Llanos AA, Marian C, McElroy J, Schneider SS, Spear SL, Troester MA, Freudenheim JL, Geyer S, Shields PG. Discovery and replication of microRNAs for breast cancer risk using
ED
genome-wide profiling. Oncotarget 2016; 7(52): 86457-86468. [6] Li Q, Liu M, Ma F, Luo Y, Cai R, Wang L, Xu N, Xu B. Circulating miR-19a and miR-205
PT
in serum may predict the sensitivity of luminal A subtype of breast cancerpatients to neoadjuvant chemotherapy with epirubicin plus paclitaxel. PLoS One 2014; 9(8): e104870.
CE
[7] Al-Khanbashi M, Caramuta S, Alajmi AM, Al-Haddabi I, Al-Riyami M, Lui WO, Al-Moundhri
AC
MS Tissue and Serum miRNA Profile in Locally Advanced Breast Cancer (LABC) in Response to Neo-Adjuvant Chemotherapy (NAC) Treatment. PLoS One 2016; 11(4): e0152032.
[8] Müller V, Gade S, Steinbach B, Loibl S, von Minckwitz G, Untch M, Schwedler K, Lübbe K, Schem C, Fasching PA, Mau C, Pantel K, Schwarzenbach H. Changes in serum levels of miR-21, miR-210, and miR-373 in HER2-positive breast cancer patients undergoing neoadjuvant therapy: a translational research project within the Geparquinto trial. Breast Cancer Res Treat 2014; 147(1): 61-8. [9] Yadav P, Mirza M, Nandi K, Jain SK, Kaza RC, Khurana N, Ray PC, Saxena A. Serum microRNA-21 expression as a prognostic and therapeutic biomarker for breast cancer patients. Tumour Biol 2016; 37(11):15275-15282. [10] Yoruker EE, Aydoğan F, Gezer U, Saip P, Dalay N. Analysis of circulating microRNAs during
ACCEPTED MANUSCRIPT adjuvant chemotherapy in patients with luminal A breast cancer. Mol Clin Oncol 2015; 3(4): 954-958. [11] Wu X, Somlo G, Yu Y, Palomares MR, Li AX, Zhou W, Chow A, Yen Y, Rossi JJ, Gao H,
T
Wang J, Yuan YC, Frankel P, Li S, Ashing-Giwa KT, Sun G, Wang Y,Smith R, Robinson K,
RI P
Ren X, Wang SE. De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locallyadvanced breast cancer. J Transl Med 2012; 10: 42.
SC
[12] Wang H, Tan G, Dong L, Cheng L, Li K, Wang Z, Luo H. Circulating miR-125b as a marker predicting chemoresistance in breast cancer. PLoS One 2012; 7(4): e34210.
MA NU
[13] Sun Y, Wang M, Lin G, Sun S, Li X, Qi J, Li J. Serum microRNA-155 as a potential biomarker to track disease in breast cancer. PLoS One 2012; 7(10): e47003. [14] Gu X, Xue JQ, Han SJ, Qian SY, Zhang WH. Circulating microRNA-451 as a predictor of resistance to neoadjuvant chemotherapy in breast cancer. Cancer Biomark 2016; 16(3):
ED
395-403.
[15] Roth C, Rack B, Muller V, Janni W, Pantel K, Schwarzenbach H. Circulating microRNAs as
PT
blood-based markers for patients with primary and metastatic breast cancer. Breast Cancer Res 2010; 12(6): R90.
CE
[16] Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG. New guidelines to evaluate the
AC
response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92(3): 205-16. [17] Huo D, Clayton WM, Yoshimatsu TF, Chen J, Olopade OI. Identification of a circulating MicroRNA signature to distinguish recurrence in breast cancer patients. Oncotarget 2016; 7(34): 55231-55248. [18] Hagrass HA, Sharaf S, Pasha HF, Tantawy EA, Mohamed RH, Kassem R. Circulating microRNAs - a new horizon in molecular diagnosis of breast cancer. Genes Cancer 2015; 6(5-6): 281-7. [19] Lee JA, Lee HY, Lee ES, Kim I, Bae JW. Prognostic Implications of MicroRNA-21 Overexpression in Invasive Ductal Carcinomas of the Breast. J Breast Cancer 2011; 14(4):
ACCEPTED MANUSCRIPT 269-75. [20] Li S, Yang X, Yang J, Zhen J, Zhang D. Serum microRNA-21 as a potential diagnostic biomarker for breast cancer: a systematic review and meta-analysis. Clin Exp Med 2016;
T
16(1): 29-35.
A, Pérez-Garcia
J, Masci
G, Corsi
RI P
[21] De Mattos-Arruda L, Bottai G, Nuciforo PG, Di Tommaso L, Giovannetti E, Peg V, Losurdo F, Cortés
J,Seoane
J, Calin
GA, Santarpia
L.
SC
MicroRNA-21 links epithelial-to-mesenchymal transition and inflammatory signals to confer resistance to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast
MA NU
cancer patients. Oncotarget 2015; 6(35): 37269-80.
[22] Bourguignon LY, Spevak CC, Wong G, Xia W, Gilad E. Hyaluronan-CD44 interaction with protein kinase C (epsilon) promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21, leading to down-regulation of the tumor suppressor protein
284(39): 26533-46.
ED
PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells. J Biol Chem 2009;
PT
[23] Gao W, Lu X, Liu L, Xu J, Feng D, Shu Y. MiRNA-21: a biomarker predictive for platinum-based adjuvant chemotherapy response in patients with non-small cell lung cancer.
CE
Cancer Biol Ther 2012; 13(5): 330-40. [24] Jin B, Liu Y, Wang H. Antagonism of miRNA-21 Sensitizes Human Gastric Cancer Cells to
AC
Paclitaxel. Cell Biochem Biophys 2015; 72: 275-82. [25] Wang B, Zhang Q. The expression and clinical significance of circulating microRNA-21 in serum of five solid tumors. J Cancer Res Clin Oncol 2012; 138: 1659-66. [26] Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DS. Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin Chem 2011; 57: 84-91. [27] Xie X, Hu Y, Xu L, Fu Y, Tu J, Zhao H, Zhang S, Hong R, Gu X. The role of miR-125b -mitochondria-caspase-3 pathway in doxorubicin resistance and therapy in human breast cancer. Tumour Biol 2015; 36: 7185-94. [28] Yang Q, Wang Y, Lu X, Zhao Z, Zhu L, Chen S, Wu Q, Chen C, Wang Z. MiR-125b regulates epithelial-mesenchymal transition via targeting Sema4C in paclitaxel-resistant breast
ACCEPTED MANUSCRIPT cancer cells. Oncotarget 2015; 6: 3268-79. [29] Vilquin P, Donini CF, Villedieu M, Grisard E, Corbo L, Bachelot T, Vendrell JA, Cohen PA.
poor prognosis in breast cancer. Breast Cancer Res 2015; 17: 13.
T
MicroRNA-125b upregulation confers aromatase inhibitor resistance and is a novel marker of
RI P
[30] Tang F, Zhang R, He Y, Zou M, Guo L, Xi T. MicroRNA-125b induces metastasis by targeting STARD13 in MCF-7 and MDA-MB-231 breast cancer cells. PLoS ONE 2012; 7:
SC
e35435.
[31] Wang HJ, Guo YQ, Tan G, Dong L, Cheng L, Li KJ, Wang ZY, Luo HF. miR-125b regulates
Biochem 2013; 114: 2248-57.
MA NU
side population in breast cancer and confers a chemoresistant phenotype. J Cell
[32] D'Angelo E, Fassan M, Maretto I, Pucciarelli S, Zanon C, Digito M, Rugge M, Nitti D, Agostini M. Serum miR-125b is a non-invasive predictive biomarker of the pre-operative
ED
chemoradiotherapy responsiveness in patients with rectal adenocarcinoma. Oncotarget 2016; 7: 28647-57.
PT
[33] Hansen TF, Carlsen AL, Heegaard NH, Sørensen FB, Jakobsen A. Changes in circulating microRNA-126 during treatment with chemotherapy and bevacizumab predictstreatment
CE
response in patients with metastatic colorectal cancer. Br J Cancer 2015; 112: 624-629. [34] Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J, Shi B. Breast cancer intrinsic subtype
AC
classification, clinical use and future trends. Am J Cancer Res 2015; 5: 2929-2943. [35] Haakensen VD, Nygaard V, Greger L, Aure MR, Fromm B, Bukholm IR, Lüders T, Chin SF, Git A, Caldas C, Kristensen VN,Brazma A, Børresen-Dale AL, Hovig E, Helland Å. Subtype-specific micro-RNA expression signatures in breast cancer progression. Int J Cancer 2016; 139: 1117-28.
ACCEPTED MANUSCRIPT
PT
Table 1 Clinicopathologic characteristic and baseline serum miRNAs expression levels of breast cancer patients
n miR-19a
miR-205
miR-451
0.70(0.31)
miR-21
miR-155
miR-373
miR-125b
0.94(0.27)
3.30(0.62)
5.41(1.84)
2.71(0.91)
0.51(1.99)
1.00(0.35)
3.28(0.55)
5.49(1.66)
2.75(0.84)
0.51(2.17)
miR-122
NU S
Characteristic
CR I
Mean of serum miRNAs expression（Standard deviation）
Age (years) 78
6.55(2.34)
5.35(1.79)
<40
40
6.70(1.99)
5.61(1.75)
Invasive ductal carcinoma
90
6.62(2.68)
5.31(1.73)
0.70(0.30)
0.96(0.31)
3.26(0.57)
5.4(1.8)
2.68(0.91)
4.97(1.92)
Others
28
6.53(2.11)
5.84(1.88)
0.66(0.21)
0.98(0.26)
3.37(0.67)
5.5(1.7)
2.86(0.81)
5.50(2.37)
Well
37
6.79(2.21)
5.23(1.48)
0.68(0.21)
1.04(0.22)
3.25(0.64)
5.55(1.49)
2.77(0.75)
5.17(2.05)
Moderate
54
6.66(2.42)
5.60(1.96)
0.71(0.32)
0.90(0.30)
3.35(0.57)
5.55(2.02)
2.70(0.95)
5.00(1.99)
MA
≥40
D
0.69(0.24)
AC
Differentiation
CE P
TE
Histological type
ACCEPTED MANUSCRIPT
6.23(1.82)
5.40(1.80)
0.68(0.31)
Ⅱ
62
6.50(2.28)
5.45(1.92)
0.70(0.32)
Ⅲ
56
6.71(2.17)
5.43(1.61)
0.69(0.24)
Positive
59
6.74(2.14)
5.35(1.59)
Negative
59
6.46(2.31)
5.53(1.95)
Positive
89
6.58(2.19)
Negative
29
Positive Negative
0.98(0.37)
3.22(0.60)
5.07(1.61)
2.68(0.97)
5.18(2.20)
3.32(0.59)
4.12(1.10) a
2.59(0.82)
3.82(1.23) a
0.96(0.28)
3.25(0.61)
6.89(1.13) a
2.87(0.94)
6.50(1.82) a
0.95(0.28)
3.25(0.60)
6.85(1.15) a
0.29(0.93)
6.42(1.84) a
0.70(0.33)
0.97(0.32)
3.33(0.60)
4.03(1.00) a
0.26(0.82)
3.76(1.19) a
5.32(1.69)a
0.67(0.24)
0.92(0.28)
3.32(0.60)
5.62(1.74)
2.69(0.89)
5.12(2.00)
6.66(2.37)
8.81(2.00)a
0.76(0.39)
1.08(0.36)
3.19(0.60)
4.88(1.78)
2.81(0.88)
5.00(2.19)
78
6.57(2.10)
5.38(1.72)
0.70(0.27)
0.92(0.27)
3.31(0.56)
5.28(1.70)
2.62(0.87)
4.89(2.06)
40
6.65(2.48)
5.56(1.89)
0.67(0.30)
1.05(0.33)
3.23(0.68)
5.75(1.89)
2.92(0.90)
5.48(1.97)
PT
27
MA
0.68(0.23)
CE P
AC
ER status
TE
D
Lymph node matastasis
0.97(0.32)
NU S
Stage
CR I
Poor
PR status
ACCEPTED MANUSCRIPT
—
—
—
—
—
—
—
—
Negative
118
6.60(2.22)
5.44(1.77)
0.69(0.28)
0.96(0.30)
3.29(0.60)
5.44(1.77)
2.72(0.89)
5.09(2.04)
Premenopause
55
6.39(1.94)
5.54(1.70)
0.68(0.23)
1.01(0.32)
3.22(0.50)
5.52(1.66)
2.70(0.84)
5.17(2.21)
Postmenopause
63
6.79(2.44)
5.35(1.85)
0.71(0.33)
0.92(0.27)
3.34(0.67)
5.37(1.88)
2.74(0.93)
5.02(1.90)
CR+PR
80
6.49(2.13)
5.33(1.66)
0.70(0.30)
0.93(0.26)
3.33(0.63)
5.33(1.66)
2.67(0.83)
4.42(1.49)a
SD+PD
38
6.84(2.42)
5.67(2.01)
0.68(0.25)
1.02(0.37)
3.21(0.52)
5.67(2.01)
2.83(1.00)
6.52(2.31)a
CR I
0
NU S
Positive
PT
HER2 stutus
TE
D
MA
Menopausal status
AC
CE P
Chemotherapy response status
ER, estrogen receptor; PR, progestrone receptor; HER2, human epidermal growth factor receptor 2. a
P < 0.01.
ACCEPTED MANUSCRIPT Table 2 Cox proportional hazards models analysis for DFS in the patients with primary breast cancer Multivariate analysis
HR
95% CI
P
Age
1.085
0.624~1.887
0.773
Histological type
0.766
0.418~1.403
SC
RI P
T
Univariate analysis
Differentiation
0.982
TNM stage
3.471
Lymph node metastasis
3.341
ER expression
1.100
95% CI
P
MA NU
0.388
0.687~1.403
0.921
1.986~6.066
<0.001
2.382
0.347~16.347
0.377
1.898~5.881
<0.001
0.912
0.107~7.747
0.933
0.592~2.046
0.763
ED 0.624
0.366~1.064
0.083
1.032
0.611~1.744
0.905
0.193
0.112~0.332
<0.001
1.291
0.610~2.731
0.504
8.390
4.500~15.644
<0.001
124.393
10.431~1483.399
<0.001
Ser-miR-125b expression at FEN
7.783
4.235~14.303
<0.001
0.209
0.015~2.833
0.239
Ser-miR-125b expression at SEN
6.598
3.673~11.850
<0.001
0.846
0.133~5.398
0.859
8.751
4.977~15.388
<0.001
51.579
13.942~190.820
<0.001
7.386
4.260~12.805
<0.001
1.696
0.794~3.623
0.173
Menopause status
CE
Chemotherapy response
PT
PR expression
HR
AC
Ser-miR-125b expression at BL
Change of Ser-miR-21 from BL to FEN Change of Ser-miR-21 from BL to SEN
ACCEPTED MANUSCRIPT Fig. 1 HE staining in breast cancer tissues. A. Invasive ductal carcinoma, ×200. B. Invasive lobular carcinoma, ×200.
RI P
T
Fig. 2 Serum levels of miRNAs in breast cancer patients and healthy controls. **P < 0.01 compared to healthy controls; #P < 0.05 compared to clinical stageⅡ.
SC
Fig. 3 Serum levels of miR-125b in responing and non-responding patients during the NCT. A. Serum levels of miR-125b in responing and non-responding patients at BL, FEN and SEN. B.
MA NU
Changes in ser-miR-125b levels from BL to FEN and from BL to SEN in responing and non-responding patients, **P < 0.01 compared to non-responding patients. Fig. 4 Serum levels of miR-21 in responing and non-responding patients during the NCT. A.
ED
Serum levels of miR-21 in responing and non-responding patients at BL, FEN and SEN. B.
PT
Changes in serum miR-21 levels from BL to FEN and from BL to SEN in responing and non-responding patients. C, D, E, F. Changes in serum miR-21 levels from BL to FEN and from
CE
BL to SEN in every patient. Black lines: decreased ser-miR-21 level; red lines: increased
AC
ser-miR-21 level. *P < 0.05 compared to BL. Fig. 5 ROC curve analysis based on serum levels of miR-125b and changes of miR-21 serum levels for the discrimination between responding and non-responding patients. A. Serum levels of miR-125b at BL. B. Serum levels of miR-125b at FEN. C. Serum levels of miR-125b at SEN. D. Changes of miR-21 serum levels from BL to FEN. E. Changes of miR-21 serum levels from BL to SEN. F. Combination miR-125b levels at BL with the changes of miR-21 levels from BL to FEN. Fig.6 Kaplan-Meier survival analysis for DFS depending on serum levels of miR-125b and changes of miR-21 serum levels. A. Serum levels of miR-125b at BL. B. Serum level of miR-125b at FEN. C. Serum level of miR-125b at SEN. D. Changes of miR-21 serum levels from BL to
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
FEN. E. Changes of miR-21 serum levels from BL to SEN.
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
Figure 1
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 2
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 3
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 4
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 5
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 6
ACCEPTED MANUSCRIPT Highlights 1. MiR-125b and miR-155 are upregulated in the serum of breast cancer patients.
T
2. Ser-iR-21 and miR-125b are associated with chemotherapy response and DFS.
AC
CE
PT
ED
MA NU
SC
RI P
3. MiR-21 and -125b are noninvasive predictive markers for NCT response and prognosis.