Abstract

INTRODUCTION

According to the International Agency for Research on Cancer, primary liver cancer is the fifth and seventh most common cancer in men and women worldwide (7.9% and 6.5% of all cancers), respectively, and the third most common cause of cancer-related death. A periodic follow-up observation for patients with chronic liver diseases, especially for cirrhotic liver using imaging modalities and tumor markers, is considered to aid in the detection of hepatocellular carcinoma (HCC) at its early stages, leading to the improvement of overall survival[1]. With the goal of earlier detection of hepatocyte transformation, several hepatocyte-specific MRI contrast agents have been investigated. Gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid (Gd-EOB-DTPA) was introduced in the clinic in 2007.

The lipophilic modification of Gd-DTPA, which is a contrast medium solely for extracellular perfusion[2], consisting of a covalent linkage with ethoxybenzyl moiety has allowed both extracellular hemodynamics and hepatocyte function to be explored using one contrast medium in MRI[3]. Gd-EOB-DTPA is actively taken up by hepatocytes, followed by excretion into bile juice. A phase I study has revealed that liver parenchyma is immediately enhanced after the injection of Gd-EOB-DTPA reaching a peak intensity 10 to 20 min after the injection, which is sustained for more than 2 h, the so-called hepatobiliary phase, in volunteers with normal functioning liver parenchyma[4]. Organic anion-transporting polypeptide 1B3 and multidrug-resistant proteins are responsible for hepatocyte uptake and excretion, respectively, of the contrast medium[5].

Imaging of hepatocytes by EOB uptake can detect a focal liver lesion with less signal intensity based on a functional deviation of hepatocytes through their transformation process[6]. Although most classical HCCs reveal less signal intensity in the hepatobiliary phase[7], it is still under debate whether non-hypervascular hepatic nodules (NHNs), which are detected in the hepatobiliary phase but not enhanced in the arterial phase (as is the case for classical HCC), are destined to result in overt HCC. This study aims to establish a prognosticator that allows clinicians to determine NHNs with a higher potential to become an overt HCC by means of grow and/or gain hypervascularity as a surrogate marker.

MATERIALS AND METHODS

RESULTS

Natural course of non-hypervascular hepatic nodules

The basic characteristics of the NHNs are summarized in supplementary Table ​Table1.1. In a median follow-up period of 504 d (IQR: 227-673 d), 68.5% or 20.5% of 73 NHNs were enlarged or reduced (including 16.4% that disappeared) by 2 mm or more in diameter, respectively, whereas the remaining 11.0% were stable in size but acquired vascularity. Representative images are shown in Figures ​Figures11 and ​and2.2. No NHN that decreased in size developed hypervascularity, whereas hypervascularity was depicted in 40.0% of the enlarged NHNs. Figure ​Figure3A3A shows the actual size distributions over the observation period. The median diameter of the 73 NHNs was 9 mm (IQR: 8-12 mm) at the initial detection. Tissue samples were obtained from 6 of 58 progressed NHNs, cases #1-3, 12-1, 14-1, 19-5, 21-1, and 22-1 allocated in supplementary Table ​Table1,1, and all of these tissues revealed a histological diagnosis of HCC with high to moderate differentiation (Figure ​(Figure3B3B).

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Figure 1

Representative gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging of non-hypervascular nodules that progressed over time. A: A non-hypervascular nodules (NHNs) approximately 10 mm in diameter was clearly detected in the hepatobiliary phase, as indicated by an arrowhead in the area ventral to the scar of radiofrequency ablation. The NHN was also detected in T1-weighted imaging (T1WI), the arterial phase and the equilibrium phase as a nodule with lower intensity. A fat deposition was unclear in the subtraction of in- and out-of-phase (In-Out) fat-suppressed T1WI. Approximately 1 year later, the NHN had grown to 20 mm in diameter and was partly enhanced in the arterial phase; B: A NHN approximately 8 mm in size was clearly visualized in the hepatobiliary phase, as indicated by an arrowhead in segment 1. The corresponding low-intensity nodule was recognized in T1WI, as well as the equilibrium phase, while a fat deposition was unclear. After approximately 5 mo, the NHN grew to 15 mm in diameter without an increase in the arterial supply.

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Figure 2

Representative gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging of non-hypervascular nodules that resolved over time. A: A non-hypervascular nodules (NHNs) was clearly detected at the surface of segment 6 in the hepatobiliary phase, as indicated by an arrowhead in the area ventrolateral to a tiny cyst. The NHN was also revealed in the arterial and equilibrium phases as a lower-intensity nodule, while neither T1-weighted imaging (T1WI) nor T2-weighted imaging visualized the NHN. A fat deposition was expected on the basis of higher intensity in the subtraction of in- and out-of-phase (In-Out) fat-suppressed T1WI. After more than 2 years, the NHN was still there, but the diameter was reduced to less than 5 mm; B: The hepatobiliary phase presented a 6-mm NHN at segment 8 in the vicinity of the diaphragmatic surface of the liver, as indicated by an arrowhead. The NHN could not be visualized in any other sequence. A subsequent gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging showed no nodular lesion in the same area after approximately 5 mo. This NHN was detected together with the other NHN that is presented in Figure ​Figure1B1B.

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Figure 3

Size distribution over time and representative microscopic images of non-hypervascular nodules that progressed without hypervascularity. A: The median diameter of 73 non-hypervascular nodules (NHNs) was 9 mm [interquartile range (IQR): 8-12 mm] at the first detection. Diameter was plotted against time to show their growth/reduction rate during the study period. NHNs were classified into 3 groups based on vascularity and size changes in the observation period: hypervascular, enlarged and reduced groups. In the hypervascular group, the vascularity in the arterial phase was confirmed at the endpoint in each case. NHNs in the enlarged group grew at least 2 mm in diameter by the end of the observation, while the diameter decreased 2 mm or more in the reduced group; B: Tissue samples were obtained from 6 of 58 progressed NHNs. Most specimens show thick (1: case #21-1 allocated in supplementary Table ​Table1)1) or thin (2: case #19-5 allocated in supplementary Table ​Table1)1) trabecular patterns of tumor cells with nuclear crowding and hypercellularity, which were diagnosed as well-differentiated hepatocellular carcinoma (HCC). One specimen (3: case #22-1 allocated in supplementary Table ​Table1)1) shows less-differentiated tumor cells with a vacuolated appearance and nuclear irregularity; it was diagnosed as moderately differentiated HCC. (H and E: Each scale bar represents 50 μm.)

Characteristics of progressive non-hypervascular hepatic nodules

At first, we compared clinicopathological factors between progressed and resolved NHNs using univariate analyses. As shown in Figure ​Figure4A,4A, NHNs were detected with a significantly higher frequency by T1WI and/or T2WI in progressed NHNs than in resolved NHNs (43.1% vs 6.7%, P = 0.01). In the arterial phase, progressed NHNs were also detected as less attenuated nodules with a significantly higher frequency than resolved NHNs (Figure ​(Figure4B,4B, 34.5% vs 6.7%, P = 0.05). When a characteristic feature was evaluated among patients and not among nodules, the patients carrying progressed NHNs were significantly older than the others (median: 75 years vs 51 years, P = 0.0004) (Figure ​(Figure4C).4C). Furthermore, serum albumin concentrations were 3.7 g/dL and 4.3 g/dL in the patients with progressed and resolved NHNs, respectively, and were significantly lower in the patients with progressed NHNs (Figure ​(Figure4D,4D, P = 0.008). Serum AFP concentration, which was expressed logarithmically, tended to be higher toward significance in patients with progressed NHNs than resolved NHNs (1.28 vs 1.10, P = 0.09).

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Figure 4

Characteristics of non-hypervascular nodules clarified by univariate analyses. Non-hypervascular nodules (NHNs) that were detected in the hepatobiliary phase of gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging were univariately characterized. NHNs were classified into 1 of 2 categories, resolved or progressed, in which NHNs decreased by 2 mm or more in diameter without hypervascularity or increased by 2 mm or more or gained an arterial supply, respectively. A: In 58 progressed NHNs, 43% were detected in T1-weighted imaging (T1WI) and/or T2-weighted imaging (T2WI) (black box), whereas only 6.7% showed positive results in the resolved NHNs by T1WI and/or T2WI imaging, leading to a significantly higher detection rate among progressed NHNs; B: In the arterial phase, progressed NHNs were detected as less attenuated nodules (black box) with significantly higher frequency than resolved NHNs were (34.5% vs 6.7%, respectively, P = 0.05); C: The median patient age in the resolved NHN group was 51 years (IQR: 38-72 years), which was significantly younger than the 75 years (IQR: 70-77.3 years) of the progressed group; D: Serum albumin concentration was significantly higher in the resolved NHN group, 4.3 g/dL (IQR: 3.6-4.3 g/dL), than in the progressed group, 3.7 g/dL (IQR: 3.3-3.9 g/dL).

DISCUSSION

A deviation of the equilibrium between the uptake and excretion of EOB due to hepatocarcinogenesis makes it possible to distinguish transforming hepatocytes as a lower-intensity nodule earlier than any other available imaging modality[8,9]. However, NHN, which is detected in the hepatobiliary phase without the characteristic hypervascularity of classical HCC, is not always destined to become an overt HCC. Akai et al[10] and Kumada et al[11] reported that 3.2% and 43.5% of NHNs, respectively, turned into hypervascular nodules in one year. Although the exact reason for the large difference between these incidences is unclear, the NHN size difference may be one of the causes, as the tumor doubling time and initial tumor diameter were correlated with each other[12,13]. The mean diameter in the Akai cohort was 8.1 mm, whereas median diameters of vascularized and non-vascularized NHNs were 20 and 14 mm, respectively, in Kumada’s report. The median diameter of the present study was 9.0 mm, and hypervascularity was confirmed in 37.0% of NHNs during a median follow-up period of 504 d. Because it is difficult to perform Gd-EOB-DTPA-MRI frequently in a large cohort, both from the patient’s point of view and because of the limited availability of hospital equipment, it is hard to define precisely when the vascularity appeared. However, it may be reasonable to assume from those 2 studies and the present study that roughly 30% of 10-mm NHNs would gain vascularity in a year. Either way, it is not likely that more than half of 10-mm NHNs progress into a classical HCC in a year. Therefore, it is critical to establish a strategy to differentiate progressive NHNs from relatively indolent ones, which would enable us to define a high-risk group and to provide a rationale for further intensive investigations, including histological evaluations.

It is important to define progressed and non-progressed features properly to determine the fate of NHNs. Previous studies solely employed hypervascularity as a hallmark for progression in NHN follow-up[10,11]. It is clear, however, that there are HCCs that lack a hypervascular phenotype. Bolondi et al[14] reported that the European Association for the Study of the Liver’s imaging criteria for the diagnosis of HCC, which require coincidental arterial hypervascularity in contrast-enhanced ultrasound and helical computed tomography, was satisfied in only 61% of 10- to 30-mm nodules detected by ultrasound in cirrhotic liver. In fact, well-differentiated HCCs were histologically diagnosed without hypervascular features in 10 and 4 NHNs from the 68 excluded and 73 included NHNs in our cohort, respectively. Because the sensitivity rather than the specificity is primary importance for a screening study of Gd-EOB-DTPA-MRI, the size increase should be incorporated into the criteria of progressive NHN irrespective of hypervascularity. Another important factor affecting the establishment of a predictive formula is how to handle NHNs that are stable in terms of vascularity and size over a follow-up period. In previous reports, stable NHNs were generally classified as non-progressive nodules. It is hard to determine, however, how much time is sufficient to judge a NHN as non-progressive when the NHN does not significantly change in size and/or vascularity. On the other hand, no NHN that decreased by 2 mm in size showed a progressed phenotype over the follow period in this study. To avoid the risk of misreading the NHN prognosis and mistakenly defining progressive NHNs as innocent nodules, it would be preferable to exclude stable NHNs from further analyses.

One limitation of our study is the relatively small number of cases. The limited case number may have resulted in an inadequate assessment of the biological variability. It seems reasonable that age, lower signal intensity in arterial phase, positivity of HBV or HCV, and DCP would affect NHN progression, based on our current understanding of the risk for HCC. For example, Shimada et al[15] reported that DCP level was significantly higher in non-cancerous parts of the liver when there were simultaneous multicentric HCCs. On the other hand, the tumor diameter was not incorporated in our NHN-progression prediction formula. While Kumada et al[11] reported that NHNs larger than 15 mm became a hypervascular nodule more frequently than smaller NHNs, the estimated regression coefficient for tumor diameter is small (-0.01) in multivariate analysis, and the tumor size was not a significant determinant even in univariate analysis in our cohort. Because the median diameter of NHNs in our cohort was 9 mm, substantially smaller than 15 mm, NHN size may not be a key determinant for NHN progression as long as the size is below a specified threshold. Smaller NHNs may have a higher potential for malignancy than larger NHNs because a strong contrast due to a larger functional deviation in EOB uptake and excretion should be required for the detection of small NHNs, for example, those less than 10 mm. Furthermore, differences in the definition of NHN progression may have caused some discrepancies with previous studies. To clarify the significance of these factors and to confirm the accuracy of our formula, it is essential to perform a prospective study using a larger cohort without any treatment except for pure regional therapies, such as resection and/or radiofrequency ablation. For convenient evaluation of NHN fate, we are providing a free application on our homepage as NHN Fate Predictor (http://www.med.niigata-u.ac.jp/in3/resident/NHN.html).

We developed a formula predicting whether NHNs will grow and/or gain vascularity in a couple of years. It is a hard task to differentiate two types of NHNs; NHNs turn into overt HCC within a short period and NHNs stay non-progressive for a substantial time. Our formula would be useful for the management of chronic liver diseases in guiding the necessity of successive examinations and treatments for NHNs detected by Gd-EOB-DTPA-MRI.

ACKNOWLEDGMENTS

We thank Mr. Tsutomu Kanazawa and all radiologic technicians of our hospital for daily excellent MRI studies based on their expertise.

COMMENTS

Footnotes

References