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1 Department of Radiology, Gifu University School of Medicine, Gifu 501-1193,
Japan.
2 Department of Radiology Services, Gifu University Hospital, 1-1 Yanagido, Gifu
501-1193, Japan.
3 Department of Surgical Oncology, Gifu University School of Medicine, Gifu
501-1193, Japan.
4 Department of Diagnostic Radiology, National Cancer Center Hospital, Tsukiji
104-0045, Japan.
Received April 2, 2004;
accepted after revision May 27, 2004.
Supported in part by the Grant for Scientific Research Expenses for Health
Labour and Welfare Programs, by the Foundation for the Promotion of Cancer
Research, and by the Research on Cancer Prevention and Health Services.
Abstract
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MATERIALS AND METHODS. The intensity of VEGF expression in HCC and in the surrounding liver was expressed as a VEGF expression index by Western blot analysis in 20 surgical specimens resected in 20 patients between March 2000 and August 2002. Findings on CT during arterial portography (n = 20) and CT hepatic arteriography (n = 17) were retrospectively evaluated to determine contrast enhancement indexes and the enhancement characteristics of HCCs and of the surrounding liver. Contrast enhancement indexes and VEGF expression indexes were correlated using a simple regression test, and enhancement characteristics and VEGF expression indexes were correlated using the Spearman's rank correlation test.
RESULTS. On CT hepatic arteriography, the contrast enhancement indexes of HCCs showed moderate inverse correlation with the VEGF expression indexes of HCCs (r = –0.57, p = 0.017) and high inverse correlation with the differences between the VEGF expression indexes of HCCs and those of livers (difference in the VEGF expression index, –0.80; p = 0.0001). The contrast enhancement index of the liver showed marginal moderate direct correlation with the VEGF expression index of the liver (0.44, p = 0.076) and high inverse correlation with the difference in the VEGF expression index (–0.71, p = 0.0013). On CT during arterial portography, the contrast enhancement indexes of HCCs showed moderate inverse correlation with the difference in the VEGF expression index (–0.51, p = 0.023). The qualitative degree of heterogeneity of hepatic artery enhancement in HCC on CT hepatic arteriography showed moderate direct correlation with the VEGF expression indexes of HCCs (0.55, p = 0.033) and high direct correlation with the difference in the VEGF expression indexes (0.73, p = 0.004).
CONCLUSION. Our results indicated that the intensity and heterogeneity of hepatic artery enhancement of HCCs on CT hepatic arteriography correlated with the degree of VEGF expression in HCCs.
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The expression of proangiogenetic factors such as VEGF appears to play an important role in the development of hepatocellular carcinoma (HCC) [4–7]. VEGF expression in HCC tissues has been considered to be associated with tumor size or histologic tumor grade [6]. Meanwhile, even in the nontumorous liver parenchyma, VEGF is expressed by sinusoidal endothelial cells and hepatocytes, whereas a modest and inconstant expression has been reported in Kupffer cells [4, 8]. Although radiologic imaging (i.e., CT or MRI) has been widely used as a tool for tumor detection, evaluations of tumor vascularity and viability, differentiations between benign and malignant tumors, and predictions of tumor growth, the relationship between radiologic findings and biomolecular angiogenetic activities in HCC and in the surrounding liver has yet to be ascertained. Investigations of this relationship may help radiologists understand radiologic imaging findings related to molecular biologic treatments in the future.
The purpose of this study was to assess the correlation between the quantitative and qualitative findings of angiographically assisted CT and the angiogenetic activities determined by Western blot analysis for VEGF in HCCs and in the surrounding nontumorous liver tissue.
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Of the 20 patients, five patients had type B viral hepatitis, and 15 had type C viral hepatitis. No patient had a history of alcohol abuse. The clinical severity and progression of cirrhosis evaluated according to the Child-Pugh classification were rated as grade A in 13 patients, grade B in six, and grade C in one. The technique of hepatectomy for tumor resection was a left lobectomy in two patients, trisegmentectomy in one, central bisegmentectomy in one, segmentectomy in two, subsegmentectomy in five, and partial resection of the liver parenchyma that was harboring tumors surrounded by noncancerous margins in nine. The number of resected tumors was one lesion in 12 patients, two lesions in two, three lesions in two, four lesions in one, and five lesions in three. If a patient had multiple lesions resected, the largest lesion and its surrounding liver were chosen to evaluate VEGF expression because multiple tumors in the same liver might influence each other in terms of angiogenic activity and cause statistical bias. Eventually, 20 HCCs ranging in size from 14 to 126 mm (mean [± SD], 49.3 ± 30.5 mm) and samples of surrounding liver were evaluated for VEGF expression. The 20 HCCs comprised two well-differentiated, 14 moderately differentiated, and four poorly differentiated tumors. The underlying liver disease documented by histopathologic study was chronic hepatitis to mild cirrhosis in five patients, moderate cirrhosis in ten, and severe cirrhosis in five.
Immunoblotting Techniques
Immediately after surgical resection, the specimen was sectioned through
the tumor center in the transaxial plane to ensure correlation with
preoperative CT. The samples of HCC and surrounding liver were obtained by
slicing thin tissue sections so that the samples were evenly obtained
throughout the HCC and the surrounding liver in the section. The tissue
samples were placed in liquid nitrogen immediately after sampling and kept at
–80°C until required for Western blotting. Approximately 5-g samples
were dissolved in 1 mL of radioimmuno-precipitation buffer (150 mmol/L of
NaCl, 50 mmol/L of tris[hydroxymethyl]aminomethane hydrochloride, pH 8.0, 0.1%
sodium dodecyl sulfate, 1% alkylaryl polyether alcohol, 1 mmol/L of
orthovanadate, 1 mmol/L of phenylmethylsulfonyl fluoride, 10 ng/mL of
leupeptin, and 10 ng/mL of aprotinin). Insoluble material was removed by
microcentrifugation at 13,000 rpm for 15 min at 4°C. Cell lysates (20 mg
of protein per lane) were subjected to 10% sodium dodecyl (lauryl)
sulfatepolyacrylamide gel electrophoresis. Proteins were transferred to
polyvinylidene difluoride membranes. After blocking membranes with
tris-buffered saline containing polysorbate 20 (10 mmol of
tris[hydroxymethyl]aminomethane hydrochloride, pH 8.0, 150 mmol of NaCl, 0.05%
polysorbate 20 [Tween 20, Cayman Chemical]) and 5% skim milk, membranes were
incubated with anti-VEGF monoclonal antibody (catalog number LC-3350-10, Lab
Vision) and then with antimouse IgG coupled with horseradish peroxidase.
Detection was performed using enhanced chemiluminescence.
We performed a control experiment to confirm the linearity (r = 0.97, p < 0.0001) between VEGF concentrations and the intensities of its corresponding electrophoretic bands (Fig. 1). VEGF expression, observed as electrophoretic bands, was quantified using image analysis software (Scion Image, Scion), which calculated the area under the histograms. Each pair of HCC samples and surrounding liver was examined using recombinant human VEGF solution (1.25 mg/mL) for calibration purposes (catalog number 2293, Genzyme-Techne). The VEGF expression index was calculated by dividing the area under the histogram for the specimen band by that of the calibration band.
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Angiographically Assisted CT Techniques
Using Seldinger's approach via a unilateral femoral artery in an
angiographic suite, we placed two 3.2-French angiographic catheters in the
superior mesenteric and hepatic arteries with a Y-shaped sheath introducer
(Twin Sheath, Medikit). When two or more hepatic arteries perfused the liver,
the artery that perfused the potentially resectable area of the liver was
selected for catheter placement. The hepatic artery catheter for CT hepatic
arteriography was placed in the common hepatic artery (18 patients) or in the
replaced right hepatic artery (two patients).
CT images were obtained using a single-detector helical CT scanner (HiSpeed Advantage SG, GE Healthcare) in the first 12 patients and an 8-MDCT scanner (Light Speed Ultra, GE Healthcare) in the remaining eight. All CT images were obtained in a craniocaudal direction with 5- to 7-mm collimation, 5–7 mm per 0.5- to 0.8-sec table speed, 120 kVp, and 200–220 mAs during a single breath-hold acquisition for 10–25 sec depending on liver size.
Subsequently, CT during arterial portography was performed after unenhanced CT. Data acquisition was started 25–35 sec after the initiation of a transcatheter injection into the superior mesenteric artery of 95 mL of nonionic contrast material containing 150 mg I/mL at 3 mL/sec, using an automated power injector (Auto-enhance A50, Nemotokyorindo).
CT hepatic arteriography was performed 10 min after CT during arterial portography. Data acquisition was started 5–10 sec after the initiation of a transcatheter hepatic artery injection of 30–40 mL of nonionic contrast material that contained 150 mg I/mL at 1.5 mL/sec using the same automated power injector. Finally, equilibrium phase CT was obtained 2–3 min after CT hepatic arteriography. Contiguous axial images 5–7 mm thick with no overlap were reconstructed from the volumetric data set.
Quantitative Image Analyses
To measure CT values in HCCs and in the surrounding liver, we drew a
circular region of interest to encompass as much of the HCC as possible and
another in the surrounding liver devoid of large hepatic vessels, focal
hepatic lesions, or calcifications. Quantitative degrees of contrast
enhancement in HCC and in the surrounding liver were expressed as contrast
enhancement indexes, which were calculated by subtracting the CT values on
unenhanced CT from those on contrast-enhanced CT.
Qualitative Image Analyses
Two experienced radiologists, blinded to the clinical information,
independently reviewed the CT findings in a retrospective manner. They
evaluated the contrast-enhanced CT with reference to unenhanced CT to
determine the degree of contrast enhancement in HCC and in the surrounding
liver using a 4-point scale: A grade of 0 stood for virtually no enhancement;
a grade of 1, for mild enhancement; a grade of 2, for moderate enhancement;
and a grade of 3, for intense enhancement. The radiologists evaluated the
degree of heterogeneity of hepatic artery contrast enhancement in HCC on CT
hepatic arteriography using a 4-point scale: A grade of 0 stood for
homogeneous enhancement; a grade of 1, for mild heterogeneity; a grade of 2,
for moderate heterogeneity; and a grade of 3, for strong heterogeneity. When a
disagreement occurred, consensus was reached by discussion.
Statistical Analysis
We correlated the sizes of HCCs with their VEGF expression indexes and with
those of the surrounding liver and with the difference in the VEGF expression
indexes, which was calculated by subtracting the VEGF expression index of the
surrounding liver from that of the HCC. We correlated the contrast enhancement
indexes of HCCs and of the surrounding liver on CT with the VEGF expression
indexes of HCCs and of the surrounding liver and with the difference in the
VEGF expression indexes. We correlated the qualitative degrees of contrast
enhancement and heterogeneity of hepatic artery contrast enhancement in HCCs
on CT with the VEGF expression indexes of HCCs and of the surrounding liver
and with the difference in the VEGF expression index. Statistical correlations
were determined using simple regression analysis for continuous data and the
Spearman's rank correlation test for categoric data. A correlation coefficient
value of up to 0.20 showed virtually no correlation, a value of
0.21–0.40 showed weak correlation, a value of 0.41–0.60 showed
moderate correlation, a value of 0.61–0.80 showed high correlation, and
a value of 0.81 or greater showed very high correlation. Interobserver
variability was assessed using the kappa test. A kappa value of up to 0.20
showed slight agreement, a value of 0.21–0.40 showed fair agreement, a
value of 0.41–0.60 showed moderate agreement, a value of 0.61–0.80
showed substantial agreement, and a value of 0.81 or greater showed almost
perfect agreement.
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The contrast enhancement indexes of HCCs and surrounding liver on CT hepatic arteriography ranged from 33 to 236 H (mean, 100.3 ± 53.1 H) and from 9 to 68 H (mean, 28.9 ± 15.7 H), respectively. The contrast enhancement indexes of HCCs and surrounding liver on CT during arterial portography ranged from 0 to 33 H (mean, 8.4 ± 7.7 H) and from 26 to 121 H (mean, 82.5 ± 23.5 H), respectively.
The degree of contrast enhancement in HCCs and in the surrounding liver on CT hepatic arteriography ranged from grades 1 to 3 (mean, 2.4 ± 0.9) and from grades 1 to 2 (mean, 1.2 ± 0.4), respectively. The degree of contrast enhancement in HCCs and in the surrounding liver on CT during arterial portography ranged from grades 0 to 1 (mean, 0.1 ± 0.3) and from grades 2 to 3 (mean, 2.7 ± 0.5), respectively. The degree of heterogeneity of hepatic artery contrast enhancement in HCCs on CT hepatic arteriography ranged from grades 0 to 3 (mean, 1.6 ± 1.1).
Tumor size showed moderate direct correlation with the VEGF expression index of HCCs (r = 0.51, p = 0.021) (Fig. 2A) and high direct correlation with the difference in the VEGF expression index (0.66, p = 0.0015) (Fig. 2B). The correlation between contrast enhancement indexes and VEGF expression intensity is summarized in Table 1. On CT hepatic arteriography, the contrast enhancement indexes of HCCs showed moderate inverse correlation with the HCC VEGF expression index (–0.57, p = 0.017) (Fig. 3A) and high inverse correlation with the difference in the VEGF expression index (–0.80, p = 0.0001) (Fig. 3B). The contrast enhancement index of the surrounding liver showed marginal moderate direct correlation with the VEGF expression index of the surrounding liver (0.44, p = 0.076) (Fig. 3C) and high inverse correlation with the difference in the VEGF expression index (–0.71, p = 0.0013) (Fig. 3D). No other significant correlation was found between the contrast enhancement index and the VEGF expression index or with the difference in the VEGF expression index. On CT during arterial portography, the contrast enhancement indexes of HCCs showed moderate inverse correlation with the difference in the VEGF expression index (–0.51, p = 0.023) (Fig. 3E), but no other significant correlation was found between the contrast enhancement index and VEGF expression index or the difference in the VEGF expression index.
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Statistical correlations between enhancement characteristics on CT and VEGF expression intensity are summarized in Table 2. Qualitatively, the degree of hepatic artery contrast enhancement in HCC on CT hepatic arteriography showed weak inverse correlation with the difference in the VEGF expression index (–0.40, p = 0.021) (Figs. 4A, 4B, 5A, 5B, 6A, 6B, 7A, 7B). The degree of heterogeneity of hepatic artery contrast enhancement in HCCs on CT hepatic arteriography showed moderate direct correlation with the HCC VEGF expression index (0.55, p = 0.033) and high direct correlation with the difference in the VEGF expression index (0.73, p = 0.004) (Figs. 4A, 4B, 5A, 5B, 6A, 6B, 7A, 7B).
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Kappa values for the two observers who performed the independent rating of images were found to range from 0.64 to 0.90 (mean, 0.77), indicating a substantial to almost perfect agreement.
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In our study, the contrast enhancement indexes of HCCs on CT hepatic arteriography were found to show moderate inverse correlation with the VEGF expression index of HCCs, which indicates that the more hypervascular an HCC is, the weaker is the VEGF expression in the tumor. It is believed that such a result reflects upregulation of VEGF expression causing the induction and suppression of VEGF expression in HCC. HCCs are often hypo- to isovascular on radiologic images during the early stage of development, typically during the stage of small well-differentiated HCC [13], and intratumoral oxygen tension at this time is believed to be low. Such a hypoxic state may induce neovascularization in HCCs, which would result in an increased supply of arterial blood and concomitant oxygen supply to the HCC and which would lead to the development of a hypervascular HCC that is typically described as a moderately to poorly differentiated HCC. However, once a hypervascular HCC has developed, VEGF gene expression may be suppressed because of the upregulation of VEGF expression. Such a scenario may be compatible with prior observations that the intensity of VEGF expression in HCCs is related to the histologic tumor grade (i.e., highest in well-differentiated HCCs and lowest in poorly differentiated HCCs) [6].
Kwak et al. [14] correlated tumor attenuation qualitatively determined on IV contrast-enhanced CT scans with immunoreactivity for the anti-VEGF antibody, which was also qualitatively determined by immunohistochemical staining. They concluded that the degree of VEGF expression in HCC is directly correlated with the degree of contrast enhancement during the hepatic artery phase. The reason that their results differ from ours in terms of the correlation between the intensity of hepatic artery tumor enhancement and VEGF expression in HCC is unclear. Eighteen (82%) of 22 patients had type B hepatitis and one (5%) had type C hepatitis in their study, whereas in our study only five (25%) of 20 patients had type B hepatitis and 15 (75%) had type C hepatitis. This substantial difference in the patient populations and in the underlying hepatic disease might reflect the contradiction in results. Additionally, we should consider the optimal correlation methodology—for example, the use of a single-detector helical CT without a bolus tracking device can lead to inconstant acquisitions of optimal hepatic artery phase images, the use of a fixed amount of iodine load per body may cause varying iodine concentrations in hepatic artery blood in individual patients, and subjective ratings of contrast enhancement on CT images and of VEGF expression may obscure statistical correlations.
Some researchers have reported that VEGF activity is not correlated with the vascularity of HCCs as determined by conventional angiography [15, 16], and others have reported that VEGF activity correlates directly with the intensity of tumor stain on angiography [5]. Although all these reports focused on the clinical significance of VEGF expression in HCC and the surrounding liver and on the evaluation of VEGF activity by means of VEGF messenger RNA quantification with Northern blotting and VEGF localization by immunohistochemical staining, the radiologic assessment of the degree of tumor staining on angiography was performed by subjective ratings using a 2- or 3-point scale. In this regard, we believe that our study technique was optimized sufficiently to clarify the relationship between the vascularity of HCCs and of the surrounding liver and their VEGF activity because contrast enhancement was quantitatively determined by a region-of-interest study on angiographically assisted CT and VEGF was determined by Western blotting.
The degree of heterogeneity of hepatic artery enhancement in HCC on CT hepatic arteriography showed moderate direct correlation with the VEGF expression index of HCC and high direct correlation with the difference in the VEGF expression index, which indicated that the more heterogeneously enhanced the HCC is, the stronger is the VEGF expression in the HCC. It has been reported that hypoxic regions of solid tumors produce powerful and directly acting angiogenic proteins such as VEGF [10].
The contrast enhancement index of the surrounding liver on CT hepatic arteriography showed moderate direct correlation with the VEGF expression index of the surrounding liver with marginal statistical significance, which suggested that the greater the hepatic artery enhancement of the surrounding liver, the stronger is VEGF expression in the liver. In their study of experimental biliary cirrhosis, Rosmorduc et al. [17] reported that there is evidence that angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia caused by liver damage. Meanwhile, El-Assal et al. [15] reported that cirrhotic livers had a significantly higher microvessel density and VEGF expression than noncirrhotic livers. The reason that in our study, the correlations between the intensity of hepatic artery enhancement and of VEGF expression were contrary between the HCCs and the surrounding liver was unclear, but we believe that this discrepancy is associated with the difference in magnitude between hepatic artery perfusion in a HCC and in the surrounding liver: The contrast enhancement indexes of HCCs ranged from 33 to 236 H (mean, 105.3 ± 51.7 H), whereas the contrast enhancement indexes of the surrounding liver ranged from 9 to 68 H (mean, 28.9 ± 15.7 H), which shows a great difference in the magnitude of the hepatic artery enhancement. Therefore, the increased oxygen supply to the HCC that was brought about by neovascularization was great enough to suppress VEGF expression via VEGF upregulation, but the increased oxygen supply in the surrounding liver might have been insufficient to cause VEGF upregulation.
The biologic interaction between the VEGF activity in a HCC and the surrounding liver has yet to be determined. As others have reported, 40–59% of HCCs have VEGF activity that is either equal to or less than that in the surrounding liver; nine (45%) of the 20 HCCs in our series had a VEGF expression index lower than that in the surrounding liver. However, even these HCCs showed outstanding arterial enhancement compared with that of the surrounding liver. Such a common radiographic observation, regardless of the reversed VEGF activity, might be attributable to the different arterial angiogenesis activity regulated by VEGF receptors in the HCC and in the surrounding liver [18] or simply to the preservation of portal vein perfusion in the local area.
Some limitations of our study should be mentioned. First, our study population was small because it was performed at a single institution. Furthermore, distribution of different types of histologic tumor grades was uneven. Multiinstitutional studies are needed to confirm our results with added statistical power. Second, although we used angiographically assisted CT to evaluate vascularity, this technique is no longer commonly used. We need to correlate the contrast enhancement of HCCs and of the surrounding liver on IV contrast-enhanced MDCT or on MRI with VEGF expression measured by Western blotting. Third, although we used Western blotting to semiquantify VEGF peptides, this technique is limited in its ability to differentiate the multiple types of VEGF peptides that exist in cell membranes, cytoplasm, and interstitial spaces. Finally, although we believe that investigations of the relationship between radiologic and molecular biologic findings will help radiologists interpret radiologic images in conjunction with molecular biologic treatments that will be clinically available in the near future, true usefulness of clinical radiologic images for this purpose has yet to be determined.
In conclusion, a moderate inverse correlation was found between the intensity of hepatic artery enhancement in HCCs on CT hepatic arteriography and the intensity of VEGF expression in HCC, and a moderate direct correlation was found between the degree of heterogeneity of hepatic artery enhancement in HCCs on CT hepatic arteriography and the intensity of VEGF expression in HCC. Our results may reflect the intensity of VEGF expression in HCC as upregulated by the increased hepatic artery blood supply to the HCC.
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