Abstract
Aim. Prediction of prognosis in hepatocellular carcinoma (HCC) is difficult. The aim of this study was to evaluate the prognostic value of serum MMP-8, -9, -13, and TIMP-1 in patients with HCC.
Methods. Pre-treatment serum samples from 134 patients with HCC were retrospectively analyzed. The serum concentration of MMP-8 was analyzed with immunofluorometric assay (IFMA), and those of MMP-9, MMP-13, and TIMP-1 were determined by enzyme-linked immunosorbent assays (ELISA). Clinical data were retrieved from patient records and survival data obtained from Statistics Finland.
Results. The overall cumulative disease-specific survival was 69% at 1 year, 50% at 2 years, and 33% at 5 years. Kaplan–Meier overall survival analysis showed that patients with low concentrations of serum MMP-8 or TIMP-1 had a statistically significantly better overall survival than patients with high concentrations of serum MMP-8 or TIMP-1 (P = 0.013 and P = 0.003). Interestingly, the overall survival in patients with high MMP-9/TIMP-1 ratio was statistically significantly better than in those patients with low MMP-9/TIMP-1 ratio (P = 0.004).
Conclusion. Our results suggest that serum MMP-8, TIMP-1, and the ratio of MMP-9/TIMP-1 might be useful adjuncts as predictors of prognosis in patients with HCC.
Key words::
Key messages
Prediction of prognosis in an individual patient with HCC is still a great challenge.
MMP-8, TIMP-1, and the MMP-9/TIMP-1 ratio are prognostic serum biomarkers in HCC.
Introduction
Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed malignancy and the third most common cause of cancer-related death in the world (Citation1). Up to 80% of the cases arise in developing countries (Citation1). The incidence in Western countries is, however, rising (Citation2,Citation3) mainly due to an increase in the prevalence of hepatitis B and C infection, increased alcohol consumption, and obesity-related fatty liver disease (Citation2).
Most HCC are detected after the onset of symptoms; the prognosis is dismal, with a 5-year survival of 0% to 10% (Citation4). However, if diagnosed at an early stage, surgical options such as liver resection, transplantation, or local ablative therapies can often be applied, increasing 5-year survival to up to 70% (Citation5,Citation6). Thus, early diagnosis is essential. Nevertheless, even for surgically treated small HCC tumors the recurrence rate is high (Citation6–8). Survival prediction in an individual HCC patient is, however, extremely difficult (Citation9). Several biomarkers have been studied to improve the prognostic accuracy, but reliable markers for prediction of HCC prognosis are not yet available (Citation10). The most widely used marker for HCC is serum alpha-fetoprotein (AFP), a fetal glycoprotein produced by the yolk sac and the fetal liver. Its prognostic value in HCC is, however, modest (Citation10). Thus, there is a need for novel biomarkers to help assess prognosis since this is a crucial step in the management of patients with HCC. Moreover, biomarkers predicting prognosis and response to therapy may facilitate development of personalized treatment of HCC.
Proteases play essential roles in a wide variety of biological destructive processes and are also associated with multiple diseases, especially in cancers (Citation11,Citation12). Matrix metalloproteinases (MMPs) are a family of structurally related but genetically distinct zinc-containing enzymes capable of degrading almost all extracellular matrix (ECM) and basement membrane components (Citation12,Citation13). They play an important role in several steps of cancer development by regulating cancer cell growth, differentiation, apoptosis, and immune surveillance (Citation14). Increased expression of certain MMPs can be detected in most human cancers, and their over-expressions are often associated with poor prognosis (Citation14). MMPs are regulated at different levels: gene expression, zymogen activation, and interaction with ECM and tissue inhibitors of metalloproteinases (TIMPs) (Citation15). TIMPs are found in most tissues and body fluids. TIMPs inhibit MMP activity, and the expression of TIMPs is regulated during development and tissue remodeling (Citation16). Additionally, TIMP-1 can promote cell proliferation (Citation17). Previous studies have implied that certain MMPs and TIMPs are involved in the malignant behavior of HCC, such as MMP-2, -9, and TIMP-1 (Citation18–21). There are, however, few available data referring to the role of MMP-8 or MMP-13 in HCC.
The objective of the current study was to evaluate the prognostic value of serum MMP-8, -9, -13, and TIMP-1 in patients with HCC.
Materials and methods
Patients
For this retrospective study, stored serum samples from 134 patients with HCC were analyzed. The patients were referred and treated at the Department of Transplantation and Liver surgery, a tertiary reference center, at Helsinki University Hospital between 1998 and 2007. Patient demographics are shown in .
Table I. Clinical characteristics of patients with hepatocellular carcinoma.
The diagnosis of HCC was confirmed histologically from core needle biopsy or examination of the surgical specimen in 124 (93%) patients. In 10 (7%) patients the diagnosis was made cytologically from fine-needle aspiration combined with two radiological modalities. The HCCs were staged according to the Barcelona Clinic Liver Cancer (BCLC) staging classification () (Citation3). Cirrhosis of the liver based on clinical evaluation, radiological findings, and/or liver biopsy was found in 63 (51%) patients. The severity of the cirrhosis was graded according to the Child–Pugh classification (). Viral hepatitis was found in seven patients, four patients had hepatitis B, and three patients hepatitis C. A history of alcohol abuse was documented in 28 patients (21%).
After clinical evaluation, 82 (61%) of the 134 patients were selected for interventional treatment. Thirty-nine (47%) patients underwent surgical resection, 26 (32%) thermoablation, 6 (7%) liver transplantation, 4 (5%) ethanol injection, 1 (1%) transarterial chemoembolization (TACE), and 6 various combinations of the above-mentioned treatments. Surgical resection was radical (R0) in all but two patients.
The cause of death and survival times was determined from death certificates obtained from Statistics Finland. The date of evaluation was defined as the starting-point of survival follow-up. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008). The study was approved by the Institutional Committee on Research Ethics.
Serum samples and assay procedures
Serum samples were obtained from the patients at the time of clinical evaluation, at a median time of 21 days (rage 1–33 days) before treatment. The samples were stored at –70°C before assay. Serum levels of MMP-9, MMP-13, and TIMP-1 were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instruction (Biotrak ELISA System; Amersham Biosciences, Buckinghamshire, UK); the detection limits were 0.6 ng/mL, 1.25 ng/mL, and 0.032 ng/mL, respectively (Citation22,Citation23). MMP-8 was detected with time-resolved immunofluorometric (IFMA) (MedixBiochemica, Kauniainen, Finland) according to the manufacturer's instructions; the detection limit was 0.08 μg/L (Citation22,Citation24). AFP was determined by a time-resolved immunofluorometric assay, and the reference range was 1–11 ng/mL (Citation25). AFP was analyzed within 2 days, and the results were retrieved from clinical records.
Analysis of data
The primary outcome was overall survival. Follow-up time was defined as the time from the date of evaluation. Molar ratios of MMPs and TIMP-1 (indicated as MMP/TIMP-1) were determined by dividing the concentrations with the corresponding molecular weights (Citation26). Comparison of continuous variables between groups was performed using the Mann–Whitney U test. A P value of less than 0.05 was considered statistically significant. In the absence of clinical information on a cut-off value differentiating low and high baseline concentration for MMP-8, -9, -13, TIMP-1, and the MMP/TIMP-1 molar ratios, samples were dichotomized on the basis of their median concentrations. For serum AFP, however, we used ≥ 400 ng/mL as cut-off, which has been used for prognosis in previous studies (Citation27). The association of each biomarker with survival was univariately estimated by comparing survival probabilities according to the Kaplan–Meier product limit method and log-rank test. Multivariate analysis was performed by Cox regression analysis. Correlations between individual markers as well as between marker levels and age, number of tumors, tumor size, Child–Pugh classification, and BCLC stage were assessed by the Spearman rank correlation test.
Results
The minimum follow-up period was 22 days or until death, while median follow-up time was 20 months (range 22 days to 109 months). The median follow-up time for patients still alive was 7 years (range 6–13 years). At the end of follow-up 111 (82%) patients had died. The cause of death was HCC in 83 patients and liver cirrhosis in 8. Causes of death are summarized in . The overall cumulative disease-specific survival was 69% at 1 year, 50% at 2 years, and 33% at 5 years. Median survival time was 26 months.
Table II. Cause of death in HCC patients.
The median serum concentrations and interquartile ranges of MMP-8, -9, TIMP-1, AFP, and the molar ratios of MMP/TIMP-1 are shown in . Measurable serum concentrations of MMP-13 were observed in only five patients with HCC.
Table III. The median serum concentrations and interquartile ranges of MMP-8, MMP-9, TIMP-1, α-fetoprotein, and the molar ratios of MMPs and TIMP-1 in patients with HCC.
Kaplan–Meier overall survival curves for patients with low and high marker concentrations are shown in . Patients with low serum MMP-8 concentration had a markedly better overall survival compared to those with high serum concentrations of MMP-8 (P = 0.013; ). The estimated median survival was 30 months (95% CI 17–43 months) versus 13 months (95% CI 7–19 months). Patients with low serum concentrations of MMP-9, on the other hand, did not have a better overall survival compared to those with high serum concentrations of MMP-9 (P = 0.330; ). Estimated median survival was 24 months (95% CI 11–38 months) versus 14 months (95% CI 8–20 months). Those patients with low serum concentrations of TIMP-1, however, had a significantly better overall survival compared to those with high serum concentrations of TIMP-1 (P = 0.003; ). Estimated median survival was 35 months (95% CI 26–44 months) versus 13 months (95% CI 8–18 months). When looking at the Kaplan–Meier survival curves and the molar ratios of MMP/TIMP-1 there were no significant difference in overall survival between patients with high or low molar ratios of MMP-8/TIMP-1 (P = 0.239; ); estimated median survival 23 months (95% CI 10–36 months versus 13 months (95% CI 2–25 months). However, there was a significant difference in overall survival between patients with high or low MMP-9/TIMP-1 ratio. Interestingly, those patients who had a high MMP-9/TIMP-1 ratio had a significantly better overall survival compared to those with low MMP-9/TIMP-1 ratio (P = 0.004; ). The estimated median survival was 28 months (95% CI 11–45 months versus 14 months (95% CI 6–21 months). There was no statistically significant difference in the overall survival between patients with high or low serum AFP concentrations (P = 0.082; ), with an estimated median survival of 24 months (95% CI 14–33 months) versus 10 months (95% CI 5–15 months).
Figure 1. Kaplan–Meier survival curves in patients with HCC. A: Low and high MMP-8 serum concentrations. B: low and high MMP-9 serum concentrations. C: Low and high TIMP-1 serum concentrations. D: Low and high molar ratios of MMP-8/TIMP-1. E: Low and high molar ratios of MMP-9/TIMP-1. F: Low and high AFP serum concentrations.
In the cohort of patients not receiving any form of interventional treatment, TIMP-1 was the only marker that could show a statistically significant difference in survival in the Kaplan–Meier survival analysis (P = 0.012); estimated median survival was 10 months (95% CI 5–16 months) versus 6 months (95% CI 4–7 months). In the cohort of patients who were surgically treated none of the markers studied could show any prognostic significance.
Cox multivariable regression analysis showed that of the markers studied serum MMP-9 and the serum MMP-9/TIMP-1 ratio were independent prognostic markers, and of the clinical parameters the therapy the patients got was an independent prognostic marker ().
Table IV. Cox multivariable regression analysis of serum MMPs and clinical parameters in relation to overall survival of HCC patients.
There was a moderate correlation between MMP-8 and the BCLC criteria (ρ = 0.299, P < 0.0001) and tumor size (ρ = 0.408, P < 0.0001). TIMP-1 correlated moderately with BCLC criteria (ρ = 0.361, P < 0.0001), and also with serum bilirubin (ρ = 0.561, P < 0.0001) and with serum albumin concentrations (ρ = 0.322, P < 0.0001). The MMP-8/TIMP-1 ratio correlated moderately with the size of the tumors (ρ = 0.322, P <0.0001).
Discussion
To the best of our knowledge, we demonstrate here for the first time that serum MMP-8, serum TIMP-1, and the serum MMP-9/TIMP-1 ratio are of prognostic value in patients with HCC.
The majority of HCC patients show at time of diagnosis advanced or unresectable disease. Even for those patients who undergo resection or are otherwise treated (e.g. TACE or radiofrequency ablation) the prognosis is poor. This is due to the recurrence rates, which are among the highest of any solid tumor, reaching 50% two years after resection (Citation28). Additionally HCCs are known to be relatively chemorefractory tumors (Citation29). Sorafenib is the only systemic therapy to date that has shown some benefit in patients with HCC, especially in patients with advanced tumors (Citation30). Systemic targeted therapy is, however, unlikely to be tolerated or of significant benefit in patients with Child–Pugh C cirrhosis or poor performance status (Citation31). In the present study none of the patients were treated with sorafenib or some other systemic drugs.
Prediction of prognosis in an individual patient with HCC is still a great challenge. Although some clinicopathological features of HCC are useful, they do not meet clinical requirements for prediction of HCC development (Citation32). There is an on-going search for predictive biomarkers of HCC, where pathological parameters, protein biomarkers, etc. are surveyed; however, specific markers are still lacking (Citation27). Currently the most widely used biomarker for detecting and monitoring HCC is still serum AFP. However, AFP is not an optimal diagnostic marker, being elevated in only about half of the patients, and its prognostic value in HCC is modest (Citation10). For prognostic purposes serum biomarkers are appealing because of the ease of obtaining samples and their relatively low cost. Thus, there is an urgent need for new serum biomarkers to complement AFP, to aid predicting prognosis and response to therapy, which may facilitate development of personalized treatment of HCC.
MMPs and their TIMP regulators are expressed at low levels in healthy tissue and body fluids, but under various pathological conditions their levels and activation are increased e.g. in inflammatory diseases and malignancies (Citation14). MMPs have gained considerable attention, because they are suggested as key regulators of tumor growth and metastasis. Nevertheless, recently certain MMPs such as MMP-8 and -9 have been demonstrated to act also in a protective manner by processing anti-inflammatory and bioactive non-matrix mediators both in inflammatory disease and malignancies (Citation33–36). In general, an over-expression of MMPs is often associated with poor survival in cancer (Citation14).
Most studies on HCC cell lines have shown that HCC cells secrete increased levels of MMPs (Citation37). By secreting MMPs, blood vessels and ECM are invaded and degraded, which eventually leads to cancer spread to distant organs as well as in the liver itself during disease progression (Citation38). MMP-9 plays a fundamental role in the regulation of tumor cell invasion, metastasis, and angiogenesis (Citation12,Citation39). MMP-9 has been shown to be secreted in human hepatoma cells and in various other cancer types including lung, colon, gastric, and breast cancer (Citation40–42) and has been associated with poor prognosis in patients with gastric carcinoma and pancreatic adenocarcinoma (Citation43). In previous studies in patients with HCC, MMP-9 expression has been shown to be a predictor of invasiveness, recurrence, and metastasis (Citation18,Citation19,Citation21,Citation40,Citation44,Citation45). Moreover, plasma MMP-9 levels were significantly elevated in patients with HCC with macroscopic portal vein invasions compared to patients with HCC without those invasions (Citation44). However, in the present study there was no difference in overall survival between HCC patients with high or low serum concentrations of MMP-9. Nevertheless, the MMP-9/TIMP-1 ratio showed a significant difference in overall survival in the present study. In the present study the overall survival was also markedly better in those patients with low serum concentrations of TIMP-1 relative to those with higher serum concentrations. This is in accordance with the study from Altadill et al. where TIMP-1 expression was associated with a shorter overall survival (Citation20). Moreover, TIMP-1 over-expression has been shown to lead to an increased migration of hepatoma cells (Citation42) and is also associated with metastasis and invasion in HCC (Citation46). Additionally to a MMP-inhibitory role TIMP-1 can also promote proliferation at wide range of cell types and even exerts anti-apoptotic functions (Citation47). Thus TIMP-1 can be regarded as a growth-factor-like mediator capable of regulating angiogenesis and inflammation (Citation47). Furthermore, it has been suggested that imbalances between MMPs and TIMPs may enhance the proteolytic burden in HCC tissues and thus promote HCC progression and metastasis (Citation48).
Among the various MMPs, the role of MMP-8 is more complex than that of the others as it has been reported to possess also antitumor activity (Citation33), and high plasma MMP-8 levels are suggested to protect against lymph node metastases in breast cancer (Citation49). On the other hand, elevated serum concentrations of MMP-8 have been observed in colorectal carcinoma (Citation50) and melanoma (Citation51). In the liver MMP-8 might have some role in resolving fibrotic hepatic tissue (Citation52). Data on MMP-8 in HCC are limited. No association has been demonstrated between MMP-8 polymorphism and HCC (Citation53). In the present study, however, there was a significant difference in overall survival between those HCC patients who had low or high serum concentrations of MMP-8.
MMP-13 is one of the few MMPs known to be expressed in the liver. Hepatic stellate cells (Citation54,Citation55), fibroblasts, Kupffer cells, and perisinusoidal cells (Citation56) express MMP-13 under various pathological and tissue-destructive conditions (Citation57). Altadill et al. reported slight expression of MMP-13 in HCC (Citation20). The median serum concentration of MMP-13 in the present study was zero: only five patients showed measurable serum concentrations of MMP-13. MMP-13 has been linked to many types of cancer (Citation13,Citation14,Citation36,Citation58). However, our present results suggest that serum MMP-13 is of no prognostic value in HCC.
In the present study HCC patients with AFP concentrations > 400 ng/mL had a less favorable overall survival compared to those patients who had serum AFP concentrations < 400 ng/mL, which is in accordance with previous studies; however, in multivariate analysis AFP was not an independent prognostic factor. A serum AFP concentration of 20 ng/mL is the most commonly used cut-off value to differentiate HCC patients form healthy adults, and levels > 400 ng/mL have been regarded as diagnostic for HCC (Citation27). Patients with serum AFP concentrations > 400 ng/mL tend to have greater tumor size, bilobar involvement, and lower median survival (Citation27,Citation59), but up to 80% of small HCC cause no increase in serum AFP concentration (Citation59). Moreover, many patients with cirrhosis and/or hepatitis have an elevated serum AFP in the absence of cancer.
Altogether, our present findings indicate that MMP-8, TIMP-1, and the MMP-9/TIMP-1 ratio are prognostic serum biomarkers in HCC. We have, however, to acknowledge some limitations in this study, such as the retrospective design and the low proportion of cirrhotic patients (51%) compared to 75% that is commonly reported. Most probably there were more Child–Pugh class A patients in the study than now recorded because the data were retrospectively collected and the presences of cirrhosis and alcohol abuse were not always appropriately recorded in the patient charts. Secondly our institution is a tertiary reference center, and most patients were pre-selected and referred to us for some sort of intervention. Our material encompassed very few patients with hepatitis C or B. A strength of the study was that in over 90% of the cases the HCC was proved by biopsy.
In conclusion, prediction of prognosis in an individual patient with HCC is still a great challenge. Our results suggest that serum MMP-8, TIMP-1, and the MMP-9/TIMP-1 ratio are promising prognostic candidates for serum biomarkers in HCC, and that they could be valuable adjuncts for identification of HCC patients with adverse prognosis. Further studies are needed to determine if they can be used as adjuncts when evaluating different treatment strategies for HCC patients.
Declaration of interest: The authors declare that they have no competing interests.
This study was supported by grants from Helsinki University Hospital Funds (EVO).
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