Introduction Hepatocellular carcinoma (HCC) is the most common type of malignancy of liver cancer, with high incidences (>500 000 cases per year) in hepatitis B virus (HBV) endemic regions, including China, Southeast Asia and sub-Sahara Africa.1 2 HCC is also highly malignant and lethal, with an overall 5-year survival rate of 5%–9% from the time of clinical diagnosis. The dismal prognosis is largely caused by late detection of the tumours when standard surgery is not operable, high recurrence rate of the malignancy and resistance to chemotherapy.3 4 Approximately 80% of patients with HCC are untreatable because of advanced tumour stages at presentation; nevertheless, curative hepatectomy can improve the 5-year survival rate to 69% if the tumour is detected earlier, particularly when the tumour is single nodule and smaller than 2 cm.5 6 Diagnosis of HCC is usually based on imaging techniques (abdominal ultrasound, MRI and contrast-enhanced CT scan showing a suspicious liver lesion), elevated serum α-fetoprotein (AFP) or optional biopsy. Advances in MRI and CT scan have greatly improved imaging of focal hypervascular mass consistent with HCC, but these procedures are costly and not readily available in developing countries. Ultrasonography can detect large lesion but fails to detect small tumour, especially in obese patients and those with underlying liver cirrhosis; and the procedures are operator-dependent, varying the diagnostic accuracy. Serum AFP has long been used as a tumour marker for HCC screening—AFP 400 ng/ml as true positive in general. The European Society for Medical Oncology (EMSO) guidelines also recommended that elevation of AFP >400 ng/ml can be used instead of fine-needle cytology for diagnosis, especially in patients with liver cirrhosis.7 Despite that, the sensitivity of AFP is only modest (sensitivity: 39%–65% and specificity: 76%–94%), leaving approximate one-third of the patients with early-stage HCC with small tumours ( 400 31 – 0 11 Alanine aminotransferase (U/l) 0.99. To ensure the reliability of the data, values that were near or below the limits of the linear quantification were removed and excluded from further analyses. miRNA concentrations were normalised to the total RNA input and expressed as the number of copies per nanogram of RNA. Statistical analysis The statistical differences of miRNA levels between tumour and adjacent non-tumour tissues were determined by paired t test with univariate p value at 400 ng/ml). The box plots show the median (horizontal bar), 25th and 75th percentiles and the whiskers of the graph show the largest and smallest values. Given the marked miRNA alteration in tumour tissues, our next question is whether certain miRNAs could be useful for detecting HCC cases, including those with low serum AFP of 100 copies/10 pg input RNA) present in the tumour tissues. Consistently, these six miRNAs (miR-15b, -21, -130b, -183, -224 and -301) were highly expressed in all HCC samples of varying serum AFP levels (figure 2B). Detection of candidate miRNAs in HCC culture supernatant Our next selection criterion is whether the miRNAs can be measured or detected in the culture supernatants from a panel of HCC cell lines. We thus assayed by qPCR the above selected miRNAs in culture media conditioned with a panel of HCC cell lines, including HepG2, Hep3B, PLC/PRF/5 and MHCC-97H. Two immortalised human hepatocyte cell lines, CL-48 and MIHA, were included as controls. All the miRNAs could be detected in the culture media, except miR-301 and miR-224, whose concentrations were beyond the detection limits and thus were excluded from further analyses. We found that miR-15b, miR-21, miR-183 and miR-130b had generally higher copy numbers in the medium of HCC cells when compared to CL-48 or MIHA (figure 3). Figure 3 TaqMan quantitative PCR assay of extracellular levels of four candidate biomarkers, miR-15b, miR-21, miR-130b and miR-183, in culture medium harvested from four hepatocellular carcinoma cell lines (HepG2, Hep3B, PLC/PRF/5 (PLC) and MHCC-97H (97H)) and two immortalised hepatocyte cell lines (CL-48 and MIHA). The assays were repeated at least twice in different samples. The error bars represent mean±SEM. Detection of miRNAs in serum samples from patients with HCC pre- and post-surgery To determine if the circulating miRNAs were derived primarily from tumours, we measured their levels in serum samples of 15 selected patients with HCC before and after surgery treatment. In agreement with the tissue data as stated above, miR-224 and miR-301 levels were very low in the sera (data not shown). For the other four miRNAs, there was a trend of reduction of miRNAs levels after surgery. As shown in figure 4, the median copies of miR-130b decreased significantly from 124.8 to 96.2 (p=0.0158) and miR-183 from 153.3 to 95.2 (p=0.0084). We also observed reductions of miR-15b and miR-21 levels after surgery, although the declination was not statistically significant. The median level of miR-15b was reduced from 177.6 to 64.1 (p=0.0637), whereas the median level of miR-21 was decreased from 221 404.4 to 100 140.5 (p=0.0648). Figure 4 Declination of serum microRNA (miRNA) markers in patients with hepatocellular carcinoma (HCC) after surgery treatment. Changes in serum levels of miR-15b, miR-21, miR-130b and miR-183 in patients with HCC (n=15) before (pre-op) and after (post-op) surgical removal of the tumours. Statistical differences were analysed using Wilcoxon signed-rank test. Table in the lower panel indicates the median level of the individual miRNAs before and after surgery. Circulating miR-15b and miR-130b classifier defines HCC cases in an independent cohort We next verified the discriminative power of these miRNAs in identifying HCC cases in an independent cohort of 30 healthy controls, 29 hepatitis B individuals and 57 patients with HCC. The levels of the four miRNAs in these serum samples were measured, and ROC analysis was performed on individual miRNAs. The cut-off of each miRNA was selected with highest Youden's Index. Among the four miRNAs, miR-130b showed the highest AUC of 0.913 (sensitivity of 87.7% and specificity of 81.4%) in separating the non-cancerous controls from those with HCCs, whereas miR-15b gave the highest sensitivity (98.3%), although its specificity (15.3%) was only modest (table 2). Table 2 Predictive performance of individual serum miRNAs and miRNA classifier (miR-15b and miR-130b) as biomarkers for detection of HCC Statistical parameters Non-cancerous controls (n=59) versus HCCs (n=57) miR-15b (≥43.485 copies/ng) miR-21 (≥73 271.020 copies/ng) miR-130b (≥97.250 copies/ng) miR-183 (≥40.100 copies/ng) miR-15b + miR-130b (≥−0.6111 sum score) Sensitivity (%) 98.25 89.47 87.72 57.89 98.25 Specificity (%) 15.25 71.19 81.36 69.49 91.53 False-positive rate (%) 84.75 28.81 18.64 30.51 8.47 False-negative rate (%) 1.75 10.53 12.28 42.11 1.75 Accuracy (%) 56.03 80.17 84.48 63.79 94.83 Positive predictive value (%) 52.83 75.00 81.97 64.71 91.80 Negative predictive value (%) 90 87.50 87.27 63.08 98.18 Positive likelihood ratio 1.159 3.105 4.705 1.898 11.593 Negative likelihood ratio 0.115 0.148 0.151 0.606 0.019 Youden's Index 0.135 0.607 0.691 0.274 0.898 Area under the receiver operating characteristic 0.485 0.865 0.913 0.661 0.981 HCC, hepatocellular carcinoma; microRNA, miRNA. Recognising the limitations of individual tests, we performed multivariate logistic regression analysis on various combinations of the miRNA biomarkers and found that the combined miR-15b and miR-130b gave the best performance. It could be a potential classifier for detecting HCC, and the formula of the classifier is as follow: − 2 . 4966 + 0 . 0532 ( copy number of miR - 130 b ) − 0 . 0210 ( copy number of miR - 15 b ). The ROC curve of the classifier has an AUC of 0.981 (figure 5A). A sum score of the classifier at −0.61109 was chosen as a cut-off, as it has the highest Youden's Index of 0.8977. At this cut-off, the classifier has enhanced sensitivity of 98.3% and specificity of 91.5% (figure 5B) when compared to the performance of individual miRNAs. Figure 5 Performance of the miR-15b and miR-130b classifier for detection of hepatocellular carcinoma (HCC) in an independent cohort. (A) Receiver operating characteristic (ROC) curve analysis of the microRNA (miRNA) classifier over patients with HCC and non-cancerous control subjects (healthy and chronic hepatitis B carriers). The miRNA classifier was derived from the logistic regression model that composed of two miRNAs, that are, miR-15b and miR-130b. (B) Dot plots of the sum score of the logistic regression model in non-cancerous control subjects (Control) and patients with HCC (HCC). The sensitivity, specificity and accuracy were calculated based on the cut-off at −0.611 that was determined by the Youden's Index. (C) ROC curve analyses of the miRNA classifier over control subjects (healthy and chronic hepatitis B individuals) and subgroup of patients with HCC with α-fetoprotein (AFP) levels >400, between 20 and 400 or 400 ng/ml (n=11) and non-cancerous controls (n=59); (2) HCCs with AFP between 20 and 400 ng/ml (n=16) and non-cancerous controls (n=59); (3) HCCs with AFP <20 ng/ml (n=30) and non-cancerous controls (n=59). We found that the classifier could accurately distinguish subjects with HCC from the controls with high sensitivity and specificity in all cases (figure 5C). For the HCC subgroup with AFP <20 ng/ml, the AUC of the classifier is 0.980, with sensitivity of 96.7% and specificity of 91.5%. Next, we evaluated the performance of miR-15b and miR-130b classifier in detecting early-stage HCC cases. As shown in figure 5D, the miRNA classifier could accurately identify 97.8% (44 out of 45) HCC cases from both tumour-node-metastasis stages I and II, whereas serum AFP (cut-off level at 20 ng/ml) could only detect 48.9% (22 out of 45) of the same cases. In short, the circulating miR-15b and miR-130b is a classifier that outperforms the serum AFP as tumour marker in detecting HCC. Discussion The present study has developed an unprecedented miRNA scoring system able to identify HCC cases that could not be detected by the conventional AFP tumour marker. Our in vitro studies also showed that the extracellular miRNAs are good indicator of the miRNA expression in cancer cells. These finding collectively implicate the potential application of circulating miRNAs as a non-invasive serological biomarker for solid malignancy like HCC. Our tumour biomarker discovery strategy is divided into three phases: (1) Exploration phase: to identify candidate markers through molecular profiling of miRNAs in matched tumour and adjacent non-tumour tissues samples from patients with HCC (n=96). (2) Selection/filtering phase: to select those miRNAs highly expressed in both tumour tissues as well as in serum samples. In addition, we used preoperative and postoperative serum samples from the same patients with HCC (n=15) to further refine those miRNAs that were likely derived from HCC tumours. After surgical removal of the HCC tumours, serum levels of these miRNAs were significantly reduced. (3) Cross-centre validation phase: to validate the miRNA biomarkers in an independent cohort (n=116), including HCC cases, hepatitis B carriers and non-malignant healthy controls from geographically distant medical centre. Herein, our findings showed that the combined miR-15b and miR-130b is a robust classifier in detecting HCC cases and outperforms AFP as tumour marker with high positive predictive value. Previous studies have suggested the potential use of circulating miRNAs in the diagnosis of HCC.20 21 The study by Qu et al 21 suggested that serum miR-16 could be used as a second-line test when the serum AFP and other markers are at normal levels. However, in our analysis, we did not find miR-16 significantly differentially expressed between tumour and adjacent normal tissues. Another study using Solexa sequencing on pooled serum samples identified miR-375 as a candidate biomarker for HBV-positive HCC, which was further validated by TaqMan miRNA qPCR assays.20 The majority of the HCC cases in our cohort were also HBV-positive, but we did not select miR-375 during our initial screen. This is because the difference was not significant when comparing the HCC tumours of low AFP level with the corresponding normal samples. Nevertheless, we indeed found miR-375 has an important role in regulating YAP oncogenic signalling pathway in HCC.22 Further investigation is under way to evaluate the application of miR-375 for identifying subtype of HCC, for which the Hippo-YAP signaling is aberrantly regulated. Limited information of miR-15b is available in HCC, and its molecular mechanisms and roles in pathogenesis remain largely unknown. Consistent with the present findings, miR-15b was recently shown to be overexpressed in HCC tumours,23 and it is in a pilot cross-sectional, phase II biomarker study for Barrett's oesophagus progression.24 Recently, miR-15b has been shown to be a direct transcriptional target of E2F and may be involved in preventing replicative stress in response to mitogenic signalling.25 On the other hand, the oncogenic role of miR-130b in hepatocarcinogenesis is prominent and is considered as a cancer stem cell miRNA in HCC. First, it has been shown highly expressed in CD133+ tumour-initiating cells in HCC, and transduction of miR-130b into CD133-negative cells could promote tumourigenesis and induce chemoresistance.26 Second, miR-130b directly targets a well-known tumour suppressor, RUNX3, and regulates expression of pro-apoptotic Bim, thereby enhancing cell viability.27 Despite of these observations, it is still unknown if miR-15b and miR-130b in circulation contribute to HCC development and tumour progression. Although the miRNA classifier gave a high positive predictive value in our HCC cohort, there are several limitations in this study. First, the classifier has yet to be validated in other ethnic populations, such as Europe and Japan in which hepatitis C virus is the major aetiology of HCC. Second, the post-surgical serum samples were in small sample size and varied at different time points. It would be imperative to test more longitudinal samples in order to justify the specific time or period that the circulating miRNAs return to basal levels. There are mounting evidences indicating that serum-based miRNAs are useful as non-invasive biomarkers for different cancer types.14 28–32 Today, circulating miRNAs in serum have been reported as potential diagnostic markers for B cell lymphoma,33 leukaemia,34 oesophageal squamous cell carcinoma,35 as well as lung,36 breast,37 colon,38 gastric,39 pancreatic,40 prostate41 and ovarian42 cancers. A handful of studies showed that miRNAs could be released into bloodstream via active secretion from diseased tissues in forms of protein-bound complex43or as membrane-bound vesicles (eg, exosomes).44 Because of the high rate of proliferation and cell lysis in tumours, non-specific passive release could also exist in cancer that contribute to the abundance of miRNAs in the bloodstream. Not only reflecting physiological and pathological changes, these circulating miRNAs could be functional and correlated with tumour progression and clinical outcomes.32 45 46 Most recently, miR-16, -21, -122 and -375 in serum were suggested as potential diagnostic markers for HCC.20 21 47 Nevertheless, their diagnostic performance has not been robustly validated in an independent cohort. Our present findings demonstrate that the circulating miR-15b and miR-130b hold promise as valuable tumour markers for detecting HCC, in which both the early-stage and low-AFP groups were covered with high sensitivity and specificity. Early detection of HCC could save many lives and enhance the quality of life in patients suffering from this lethal malignancy. Supplementary Material Supporting Statement Author's manuscript Reviewer comments
