Exosomal miR-34s panel as potential novel diagnostic and prognostic biomarker in patients with hepatoblastoma

Journal of Pediatric Surgery 52 (2017) 618–624

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Exosomal miR-34s panel as potential novel diagnostic and prognostic biomarker in patients with hepatoblastoma☆,☆☆ Chenwei Jiao a, Xiaohu Jiao b, Anzhi Zhu c, Juntao Ge a, Xiaoqing Xu d,⁎ a

Department of Pediatric Surgery, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China Department of Surgery, Baoji Hospital affiliated to Xi'an Medical University, Baoji, China Department of Pediatric Surgery of The Second People's Hospital of Liaocheng city, Linqing, China d Department of Radiation Oncology, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, China b c

a r t i c l e

i n f o

Article history: Received 16 June 2016 Received in revised form 21 September 2016 Accepted 22 September 2016 Key words: Exosome Hepatoblastoma miR-34s

a b s t r a c t Purpose: The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of patients with HB in a large Asian group and explore the prognostic value of the exosomal miRNA-34s panel compared with other risk factors. Methods: We retrospectively reviewed 89 children with HB. Among these patients, 63 patients were included as training group to build the diagnostic model for HB. 26 patients were defined as the validation group. The expressions of miRNA-34s were detected by real-time PCR. The comparison of diagnostic and prognostic performance of serum exosomal miRNA-34s was measured using the area under ROC curve (AUC). Results: For patients in the training group, expression of miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with control group in serum exosomes. Between HB training group and the control group, exosomal miRNA-34a, miRNA-34b and miRNA-34c had no significant differences compared with the AFP level in diagnosing HB. The performance of the exosomal miRNA-34s panel in differentiating the HB training group from the control group was superior to the AFP level. The value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in both training group and validation group. Conclusions: In this study, we found that the expression of exosomal miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with the control group, and we confirmed the exosomal miRNA-34s panel could be defined as a diagnostic and prognostic biomarker for patients with HB. Level of evidence: Level II. Type of study: Retrospective Study. © 2017 Elsevier Inc. All rights reserved.

Hepatoblastoma (HB) is the most common primary malignant tumor of the liver in young children, accounting for approximately 0.8% to 2.0% of all pediatric malignancies and 80% of all hepatic malignant tumors [1,2]. Although currently patients are routinely treated with combinatorial chemotherapy, curative resection of the primary tumor, and sometimes radiotherapy [3], a significant proportion of HB patients still have a risk of local relapse or distant metastasis even

☆ Author contributions: Jiao Chenwei and Xu Xiaoqing designed research; Jiao Xiaohu, Zhu Anzhi and Ge Juntao conducted acquisition of data; Jiao Chenwei performed research and statistical analysis; Jiao Chenwei and Xu Xiaoqing wrote the paper; Xu Xiaoqing conducted a critical revision of the manuscript. ☆☆ Conflict of interest: The authors who have taken part in this study declared that they have nothing to disclose regarding funding or conflict of interest with respect to this manuscript. ⁎ Corresponding author at: Department of Radiation Oncology, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences, No.440 Jiyan road, Jinan, Shangdong, China, 250117. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.jpedsurg.2016.09.070 0022-3468/© 2017 Elsevier Inc. All rights reserved.

after surgery and intensive chemotherapy. The main reason is that most children with HB are found at PRETEXT(Pre Treatment Extent of Disease staging System) III or IV at diagnosis [4,5]. Lack of an effective means of early diagnosis is a pivotal reason contributing to the relative worse prognosis for patients with HB. Furthermore, searching for effective biomarkers to predict prognosis would be helpful for risk stratification of HB patients. MicroRNAs (miRNAs), existing naturally as the most biologically stable nucleic acid molecule with only about 19–23 nucleotides, act as fine-tuning regulators of gene expression at posttranscriptional level through a complicated miRNA–mRNA interaction [6]. MiRNAs are emerging as a new class of regulatory molecules involved in numerous biologic processes [7]. Accumulating evidence indicated that circulating miRNAs were involved in several pathophysiological processes and related to cancer [8,9]. Numerous reports showed that the circulating miRNAs were applied as novel noninvasive biomarkers for cancers, such as colorectal cancer [10], breast cancer [11,12] and renal cell carcinoma [13].

C. Jiao et al. / Journal of Pediatric Surgery 52 (2017) 618–624

Exosomes, which are released into circulation from all cell types, are lipid bilayer cup-shaped nanovesicles 40–100 nm in diameter and provide membrane protection for inclusive RNAs and proteins [14,15]. Large amounts of exosomes can be secreted by tumor cells which were stimulated owing to the influence of hypoxia, internal environmental changes and other factors. These exosomes are not easily degradable in either intercellular space or peripheral blood owing to the protection by plasma membranes. Until now, emerging studies have suggested that tumor-derived exosomes quantitatively predominate in peripheral blood and exosome-mediated miRNA transduction plays a pivotal role in the dialogue between human tumors and their microenvironment [16]. MiRNA-34s (miR-34s) had significant role in different human cancers and almost all members of the miRNA-34 family were frequently repressed in tumors compared with normal tissue of patients in a variety of tumor types [17–19]. The miRNA-34 family consists of three members: miRNA-34a, miRNA-34b, and miRNA-34c. MiRNA-34a is encoded in the second exon of a gene located on chromosome 1p36.22, whereas miRNA-34b and miRNA-34c share a common host gene located on chromosome 11q23.1. Many studies showed that miRNA-34s family members are significant downstream effectors of p53 and contributed to carcinogenesis through different mechanisms. MiRNA-34a had been found to be significantly downregulated in metastatic breast cancer tissues when compared to nonmetastatic breast cancer. Thus, the expression level of miRNA-34a might be useful as a biomarker of metastasis [20]. MiRNA-34b/c had been determined to play a crucial role in pathogenesis of small cell carcinoma of lung. MiRNA-34b had also been revealed to correlate with invasive capacity of the small cell carcinoma of lung cell [21]. MiRNA-34c had been shown to correlate with invasive capacity of human pancreatic cancer cell. With respect to HB, our previous study showed that miRNA-34s were deregulated in tumor tissues compared with corresponding noncancerous tissue samples. We also confirmed that combined low miRNA-34s are an independent prognostic factor related with HB [22]. Based on the crucial roles of miRNA-34s family and the exosomes in various cancers and our previous results, we aim to explore the diagnostic and prognostic values of serum exosomal miRNA-34s in children with hepatoblastoma in this study. 1. Materials and methods 1.1. Patients and blood samples We prospectively collected data from consecutive patients with primary HB who underwent a curative liver resection in Shandong Provincial Hospital affiliated to Shandong University or Shandong Cancer Hospital affiliated to Shandong University between February 2007 and March 2015. HB diagnosis was based on WHO criteria. Tumor staging was determined according to the 7th edition of the tumor–node–metastasis (TNM) classification of the International Union against Cancer. Ultimately, 89 patients enrolled in this study. We used logistic regression model to build a combined factor for predicting prognosis of children with HB in this study. And we need to test this model in a validation group. Therefore, among these patients, 63 patients were included as training group to build the prognostic model for HB. 26 patients were defined as the validation group. Moreover, blood samples were also collected from all patients and 63 healthy children with matching ages and genders to the HB training group. Written consents were obtained from all subjects prior to the recruitment. The study protocol was approved by the Research Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University. The clinical characteristics of the subjects are listed in Table 1. 1.2. Extraction of exosomes from peripheral blood Whole blood was centrifuged at 3000 ×g for 15 min to remove cells or cell debris; the supernatant liquid was then placed into a centrifuge

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tube, added with 63 μl of ExoQuick reagent per 250 μl of serum and allowed to stand at 4 °C for 30 min. In a 4 °C environment, the mixture was centrifuged at 1500 × g for 30 min (exosomes precipitated at the bottom of the centrifuge tube). Supernatant was aspirated completely and centrifuged at 1500 ×g, 4 °C for 5 min. Supernatant was aspirated completely (during which there should be no shaking of the centrifuge tube), completely dissolved and precipitated with 20 μl of 1× PBS and stored at −20 °C. 1.3. Extraction of total RNA from serum exosomes 200 μl of sample was dispensed and added with 200 μl of 2× denaturing solution; then the mixture was placed in ice for 5 min. Next, an equivalent volume of acid-phenol:chloroform was added, test tube was shaken for 50 s, and the mixture was centrifuged at 10,000 ×g for 5 min. The supernatant was transferred to a fresh tube, elution solution was preheated, supernatant was added with a 1.25-fold volume of absolute ethanol, and the mixture was placed into a Filter cartridge and centrifuged at 10,000 ×g for 15 s, then base solution was discarded. 700 μl of miRNA wash solution 1 was added onto the Filter cartridge, centrifuged at 10,000 × g for 15 s, then base solution was discarded; 500 μl of wash solution 2/3 was added, centrifuged at 10,000 × g for 15 s, then base solution was discarded (repeated twice). The Filter cartridge was placed into a fresh tube, added with 35 μl of 95 °C elution solution, centrifuged at 10,000 ×g for 1 min, then the eluate was collected and stored at −70 °C. The extracted genomic DNAs were tested for purity and content with UV–Vis spectrophotometer. 1.4. Quantification of miRNAs(miRNA-34a, miRNA-34b and miRNA-34c expressions) in serum exosomes of patients with HB and the control group Total RNA was isolated from the exosomal pellets, the exosomedepleted supernatant, and whole serum using isothiocyanate-phenol/ chloroform extraction procedures. We typically extracted 2 μg to 9 μg of total RNA, and OD260/280 ratios typically ranged from 1.8 to 2.0, indicating high RNA purity. 10 ng of total RNA was used for each miRNA quantification. miRNA detection was performed on the Eppendorf Mastercycler EP Gradient S (Eppendorf, Germany) using commercial assays (TaqMan microRNA assays; Applied Biosystems, Foster City, CA, USA) for miRNAs. Relative quantification was calculated using 2 −ΔΔCt, where Ct is cycle threshold. Normalization was performed with universal small nuclear RNA U6 (RNU6B). Each sample was examined in triplicate, and the mean values were calculated. Meanwhile, exosomal miRNA-34s of each patient were tested at diagnosis, after chemo, prior to resection and after resection. mRNA levels in tumor samples/ nontumorous samples of 0.5-fold were defined as underexpression of the gene, whereas a ratio of 2.0-fold was defined as overexpression. 1.5. Diagnosis and treatment After a detailed history and a thorough physical examination, blood was collected for liver function test and tumor markers. Contrastenhanced computerized tomography (CT) and/or magnetic resonance imaging (MRI) was used to confirm the PRETEXT staging at diagnosis. Patient with PRETEXT stage III and IV tumors determined to be surgically unresectable at diagnosis achieved radiographic resectability after neoadjuvant chemotherapy. The chemotherapy regimens were platinum-based chemotherapy and included different regimens to different patients, involving vincristine, fluorouracil, doxorubicin and cyclophosphamide. Patients were evaluated for surgical resectability after 2, 4, 5, 6 cycles, of neoadjuvant chemotherapy. In addition, there is evidence that resistance to chemotherapy is more common after four or five chemotherapy cycles; therefore, six chemotherapy cycles were the maximum with similar chemotherapy regimens. Liver resection was carried out, taking note of the tumor diameter, location, tumor extension and estimated volume of the remaining

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Table 1 Patient and tumor characteristics. Variable

Control group

HB training group

HB validation group

No. age in months ≤6 months N6 months and b1 year N1 year Sex Female Male Diameter (cm) AFP at diagnosis (ng/ml) AFP = b10,000 (ng/ml) AFP N10,000 (ng/ml) Platelets (109/L) b600 × 109/L ≥600 × 109/L Tumor characteristics Histology (main component) Epithelial type Nonepithelial type PRETEXT Stage I II III IV Involvement of vascular Vena cava Portal vein Metastases yes no

63

63

26

8 (13%) 15 (24%) 40 (63%)

10 (16%) 16 (25%) 37 (59%)

6 (23%) 10 (39%) 10 (38%)

30 (48%) 33 (52%) – 241.4 ± 123.6 52 (83%) 11 (17%) 227.3 ± 32.2 63 0

28 (44%) 35 (56%) 6.8 ± 4.1 13,250.1 ± 2195.6 29 (46%) 34 (54%) 393.2 ± 185.6 24 (38%) 39 (62%)

9 (35%) 17 (65%) 7.2 ± 2.8 24,526.7 ± 2719.2 12 (46%) 14 (54%) 376.9 ± 138.6 11 (42%) 15 (58%)

– –

16 (25%) 47 (75%)

8 (31%) 18 (69%)

– – – –

7 (11%) 10 (16%) 20 (32%) 26 (41%)

2 (8%) 2 (8%) 9 (34%) 13 (50%)

– –

7 (11%) 9 (14%)

3 (12%) 5 (19%)

– –

14 (22%) 49 (78%)

7 (27%) 19 (73%)

P values 0.300

0.530

0.325 0.001

0.001

0.604

0.684

0.770

0.888

Abbreviations: AFP, alpha-fetoprotein; PRETEXT, Pre Treatment Extent of Disease.

liver. Liver resection was performed following Couinaud's segments, sectors and hemilivers. 1.6. Statistical analysis Continuous variables were expressed as mean ± SD (standard deviation) and compared using a two-tailed unpaired Student's t test; categorical variables were compared using χ 2 or Fisher analysis. The diagnostic performance of serum exosomal miRNA-34s was measured using the area under ROC curve (AUC). AUCs were also applied to compare exosomal miRNA-34s with AFP level using the Hanley and McNeil method [23]. The exosomal miRNA-34s panel was established based on the logistic regression model for the differentiation between the HB group and the control group. Meanwhile, calculation formula for exosomal miRNA-34s panel was obtained: logit(P = HB) = 3.2 − 1.821 * miR-34a − 2.496 * miR-34b − 3.334 * miR-34c. Furthermore, the exosomal miRNA-34s panel was used as a variable in a Cox proportional hazards regression [24] to evaluate of Event-free Survival (EFS) as the primary end-point for patients with HB. AUCs were further used to compare the predictive performance for EFS among independent prognostic factors. Statistical analysis was conducted with the SPSS for Windows version 18.0 release (SPSS, Inc., Chicago, IL) and ROC curve analysis was computed using MedCalcV.11.0.3.0 (MedCalc software, Mariakerke, Belgium). A value of P b 0.05 was considered significant in all the analysis. 2. Results 2.1. Patients characteristics 89 children divided into training and validation groups were recruited into this study. The median follow-up was 4.5 years (range 4.2 months–7.9 years). The baseline characteristics of patients at

diagnosis and the healthy children from control group were summarized in Table 1. Overall, among the three groups, the gender distribution was roughly equal (M:F = 1.1:1.25:1.8). The AFP levels of HB groups were significantly higher than those of control group (p b 0.001). The PRETEXT status of most patients was PRETEXT III and PRETEXT IV in both HB training group (73%) and validation group (84.6%), respectively. Most patients had no vascular invasion and no metastases in the HB training group (74.6%, 77.8%) and validation group (69.3%, 73.1%), respectively. 2.2. Comparing expression levels of miRNA-34a, miRNA-34b and miRNA34c in serum exosomes between HB and control groups Expression of miR-34a miR-34b and miR-34c in serum exosomes was significantly lower in patients with HB compared with control group (miR-34a: Fig. 1A, B; miR-34b: Fig. 1C, D; miR-34c: Fig. 1E, F). 2.3. Comparing diagnostic performance for patients with HB between miRNA-34s in exosomes and AFP levels Between patients in HB training group and the control group, exosomal miRNA-34a had no significant difference compared with the AFP level in diagnosing HB. The AUC of exosomal miRNA-34a was 0.831 (95% CI, 0.754 to 0.892), which was as large as that of AFP level (0.835, 95% CI, 0.758 to 0.895, Fig. 2A); exosomal miRNA-34b had no significant difference compared with the AFP level in diagnosing HB. The AUC of exosomal miRNA-34b was 0.813 (95% CI, 0.734 to 0.877), which was as large as that of AFP level (0.835, 95% CI, 0.758 to 0.895, Fig. 2B); exosomal miRNA-34c had no significant difference compared with the AFP level in diagnosing HB. The AUC of exosomal miRNA-34c was 0.837 (95% CI, 0.760 to 0.896), which was as large as that of AFP level (0.835, 95% CI, 0.758 to 0.895, Fig. 2C).

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Fig. 1. The differential expression of exosomal miRNA-34s in the HB and control groups. A, B: the comparison of miRNA-34a expression level in HB and control groups; C, D: the comparison of miRNA-34b expression level in HB and control groups; E, F: the comparison of miRNA-34c expression level in HB and control groups.

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Fig. 2. Comparison of diagnostic significance for patients with HB between exosomal miRNA-34s and AFP levels. A: AUC comparison between exosomal miRNA-34a and AFP level; B: AUC comparison between exosomal miRNA-34b and AFP level; C: AUC comparison between exosomal miRNA-34c and AFP level; D: AUC comparison between the exosomal miRNA-34s panel and AFP level.

2.4. Multivariate logistic regression analysis to establish the exosomal miRNA-34s panel as a diagnostic marker for patients with HB in the training group Considering that the expressions of exosomal miRNA-34a, miRNA34b and miRNA-34c had excellent diagnostic sensitivity and specificity for HB, we performed multivariate logistic regression analysis on these variables and combining them as exosomal miRNA-34s panel to diagnose patients with HB (Table 2). The performance of the exosomal Table 2 Logistic analysis of miRNA-34s and the diagnostic performance of patients with HB in training group compared with control group. MicroRNA group

miRNA-34a miRNA-34b miRNA-34c

AUC

0.831 0.813 0.837

Univariate

Multivariate

P value

HR

95%CI

P value

0.001 0.001 0.001

0.162 0.082 0.036

0.071–0.371 0.023–0.296 0.004–0.342

0.001 0.001 0.004

NOTE: Abbreviations: CI, confidence interval; AUC, area under the receiver operating characteristic curve; HB, hepatoblastoma. logit(P = HB) = 3.2 − 1.821 * miR-34a − 2.496 * miR-34b − 3.334 * miR-34c.

miRNA-34s panel in differentiating the HB training group from the control group was also evaluated and we found that it had significant difference compared with the AFP level in diagnosing HB (AUC 0.923; 95% CI, 0.862 to 0.963, p = 0.0402, Fig. 2D).

2.5. Prognostic value of the exosomal miRNA-34s panel compared with other conventional predictors for patients with HB in the training group Cox proportional hazards models were then used to quantify the prognostic significance of risk factors after multivariable adjustment. A multivariable analysis was performed to assess the factors that demonstrated significant effects as in univariate analysis. After adjusting for competing risk factors, we identified that AFP level, tumor metastases, vascular invasion, PRETEXT stage and exosomal miRNA-34s were associated with a worse prognosis in the multivariable adjusted analysis (Table 3). Meanwhile, we performed ROC curves to compare the prognostic performance of these independent risk factors for patients with HB. We found that the value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in training group (Fig. 3A).

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Table 3 Cox proportional hazard regression analyses in the training group. Variable

age in months Male AFP N 10,000 (ng/ml) PLT ≥ 600 × 109/L Nonepithelial type Vascular invasion Metastases: Yes/No PRETEXT IV exosomal miRNA-34s panel

Univariate

Multivariate

HR

95% CI

P value

1.021 1.105 1.758 1.169 1.295 1.687 2.602 1.8201 2.271

0.911–1.017 0.864–1.731 1.385–2.353 0.935–1.438 0.962–1.728 1.295–2.198 1.963–3.487 1.393–2.389 1.711–3.432

0.725 0.369 b0.001 0.259 0.068 0.001 b0.001 b0.001 b0.001

HR

95% CI

P value

1.659

1.291–1.903

0.001

1.454 1.638 1.958 1.861

1.243–1.817 1.122–2.357 1.424–2.644 1.421–2.494

0.012 0.008 b0.001 b0.001

Abbreviations: CI, confidence interval; PLT: blood platelet; AFP, alpha-fetoprotein; PRETEXT, Pre Treatment Extent of Disease.

2.6. Validation of predictive performance of the exosomal miRNA-34s panel According to the exosomal miRNA-34s panel established in the training group, we performed ROC curves to compare the prognostic performance of the exosomal miRNA-34s panel with other independent risk factors verified in the training group. We found that the exosomal miRNA-34s panel had significantly larger AUC than those of other predictors (Fig. 3B). The value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in validation group. 3. Discussion The outcomes for children with HB have improved markedly over the last 30 years owing to the introduction of effective chemotherapy regimens and advances in surgical treatment [25]. Many Asian studies, however, included a high proportion of patients with advanced disease, including those categorized as PRETEXT III or IV, and patients with primary refractory disease and metastatic disease. Early diagnosis could be “a sense of urgency” for improving the prognosis and reducing the burden of patients with HB. Many studies showed that exosomes present in the urine, pleuroperitoneal fluid and exosome had pleiotropic biological functions, including antigenpresenting, intracellular communication and transmission of signals and

transferring of RNAs and miRNAs [26,27]. Similarly, miRNAs were repeatedly reported as diagnostic indicator and prognostic factor in various cancers, while few studies reported the significance of the exosomal miRNAs in cancer diagnosis and prognosis predicting. Conventionally serum AFP level was used as an important diagnostic mean for patients with HB. Moreover, AFP level had been reported to be associated with survival outcomes in HB patients [28,29]. However, the sensitivity and specificity were not satisfied owing to the various sources of AFP from the fetus. With respect to the influence of AFP on prognosis, the appropriate cutoff of AFP levels to use in predicting prognosis had been a matter of some debate. AFP levels b 100 ng/mL and N1,000,000 ng/mL had been reported to be associated with poor outcome in some studies [29] but not in others [30]. The liver tumor study group of the German Society for Pediatric Oncology and Hematology (Gesellschaft fu¨r Pa¨diatrische Onkologie und Ha¨matologie, GPOH) highlighted the possible negative influence on survival outcomes of an AFP level greater than 10 6ng/mL [31], but no other report had supported this original observation. In this study, we investigated that the exosomal miRNA-34s were not inferior to AFP levels in diagnosing HB, respectively. However, after being established by logistic regression, our results demonstrated that the exosomal miRNA-34s panel was superior to AFP levels in diagnosing HB. On the basis of the ROC curves analysis and the previous studies [32], we confirmed that the serum AFP level independently impacted the postoperative survival in patients

Fig. 3. The performance of exosomal miRNA-34s panel in prognostic prediction compared with other independent risk factors among patients with HB in training group (A) and validation group (B).

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with HB. However, the predictive value of AFP level was inferior to the exosomal miRNA-34s panel in HB prognosis. Accumulating evidence has demonstrated that miRNA-34s are involved in tumor initiation, progression and metastasis [33,34]. The discovery of miRNA-34s as p53-regulated miRNAs prompted many researchers to investigate their role in cancer. All members of miRNA34 family were shown to suppress tumor growth and metastasis by inhibiting processes that promote cancer development, including cell cycle, EMT, metastasis, and stemness, and by promoting processes that inhibit carcinogenesis, such as apoptosis and senescence. MiRNA-34s regulate these processes through downregulation of their target mRNAs, which have been validated [35,36]. It had been shown that miRNA-34a inhibited the cell cycle and proliferation of lymphoma cells by repressing its target MAP2K1 (MEK1), which is a central component of MEK/ERK signaling [37]. Furthermore, it was reported that ectopic expression of all members of the miRNA-34 family induces cell cycle arrest in a variety of cancer cell lines by repression of their targets Cyclins D1 and E2, and the cyclin-dependent kinases CDK4 and CDK6 [38]; studies confirmed the proapoptotic functions of miRNA-34a in various cancer entities and several antiapoptotic genes were identified as miRNA-34 targets [39]. Inhibition of miRNA-34a protected cells to some extent from the DNA damage-induced apoptosis in wild-type p53-expressing cells, suggesting that miRNA-34a was at least in part required for p53-induced apoptosis [40]. Bcl-2, the prominent regulator of apoptosis, was identified as a direct target of miRNA-34 and proapoptotic functions of miRNA-34 are presumably mediated mainly via repression of Bcl-2 [41]. These significant characteristics of miRNA34s family and the close connection between exosomes and miRNAs contained in exosomes promoted the development of this study. The present study has several limitations and strengths. Firstly, this is a retrospective study and the sample size is too small in this study. Secondly, the potential pathogenesis of the exosomal miRNA-34s was not involved in this study and further studies are needed. However, this study provides novel means of HB diagnosis except for AFP level and imaging findings. Moreover, the exosomal miRNA-34s are closely related the HB prognosis. In conclusion, we found that expression of exosomal miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with the control group, and we confirmed the exosomal miRNA-34s panel could be defined as a diagnostic and prognostic biomarker for patients with HB. References [1] Otte JB, Pritchard J, Aronson DC, et al. Liver transplantation for hepatoblastoma: results from the International Society of Pediatric Oncology (SIOP) study SIOPEL-1 and review of the world experience. Pediatr Blood Cancer 2004;42:74–83. [2] Tiao GM, Bobey N, Allen S, et al. The current management of hepatoblastoma: a combination of chemotherapy, conventional resection, and liver transplantation. J Pediatr 2005;146:204–11. [3] Horton JD, Lee S, Brown SR, et al. Survival trends in children with hepatoblastoma. Pediatr Surg Int 2009;25:407–12. [4] Maibach R, Roebuck D, Brugieres L, et al. Prognostic stratification for children with hepatoblastoma: the SIOPEL experience. Eur J Cancer 2012;48:1543–9. [5] Qiao GL, Li L, Cheng W, et al. Predictors of survival after resection of children with hepatoblastoma: a single Asian center experience. Eur J Surg Oncol 2014;40: 1533–9. [6] Naidu S, Magee P, Garofalo M. MiRNA-based therapeutic intervention of cancer. J Hematol Oncol 2015;8:68. [7] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136: 215–33. [8] Wang K, Zhang S, Marzolf B, et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A 2009;106:4402–7. [9] Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 2011;108:5003–8.

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