Arterial Blood Supply of Hepatocellular Carcinoma and Histologic Grading: Radiologic Pathologic Correlation

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1 sayama et al. rterial lood Supply of HCC and Histologic Grading Hepatobiliary Imaging Original Research Yoshiki sayama 1,2 Kengo Yoshimitsu 1 Yunosuke Nishihara 3 Hiroyuki Irie 1 Shinichi ishima 3 kinobu Taketomi 4 Hiroshi Honda 1 sayama Y, Yoshimitsu K, Nishihara Y, et al. Keywords: CT, hemodynamics, hepatocellular carcinoma, liver disease, proliferative activity, unpaired artery, vascular imaging DOI: /JR Received February 23, 2007; accepted after revision ugust 7, Department of Clinical Radiology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan. 2 Present address: Department of Radiology, University of Iowa Hospitals and Clinics, 200 Hawkins Dr., Iowa City, I ddress correspondence to Y. sayama. 3 Department of natomic Pathology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan. 4 Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan. WE This is a Web exclusive article. JR 2008; 190:W28 W X/08/1901 W28 merican Roentgen Ray Society rterial lood Supply of Hepatocellular Carcinoma and Histologic Grading: Radiologic Pathologic Correlation OJECTIVE. The objective of our study was to clarify the sequential changes of arterial blood supply during the development of hepatocellular carcinoma (HCC) with an emphasis on its late stage. MTERILS ND METHODS. Sixty HCC nodules were confirmed at pathology in 59 patients who had undergone CT hepatic arteriography and CT during arterioportography (CTP). Lesions were classified into one of the four groups: group 1, nodules that appeared to show preserved portal perfusion on CTP and showed hypo- or isoattenuation on CT hepatic arteriography; group 2, very hyperattenuating on CT hepatic arteriography; group 3, hyperattenuating on CT hepatic arteriography; and group 4, hypo- or isoattenuating on CT hepatic arteriography. s 2, 3, and 4 showed the portal perfusion defect on CTP. These hemodynamic patterns were compared among different histologic grades. We also examined the number of unpaired arteries and the Ki-67 labeling index of the tumor pathologically. RESULTS. The numbers of lesions in each group were as follows for groups 1, 2, 3, and 4, respectively: 17, one, two, and 0 well-differentiated HCCs; 0, 10, nine, and one moderately differentiated HCC; and 0, three, 12, and five poorly differentiated HCCs. The lesions showed significantly different hemodynamic patterns among different histologic grades (Cramer s V = , p < ). The number of unpaired arteries showed a strong correlation with Ki- 67 labeling index in well-differentiated HCC and moderately differentiated HCC (rho = 0.673, p < ) and a moderate inverse correlation with Ki-67 labeling index in poorly differentiated HCC (rho = 0.540, p = ). CONCLUSION. In the late stage of HCC development, the arterial blood supply significantly decreases as the histologic grade progresses. epatocellular carcinoma (HCC) is H one of the most common malignancies in many parts of the world [1]. Recent advances in imaging have enabled researchers to evaluate the hemodynamics of a hepatocellular lesion in particular, dysplastic nodules or early HCCs using CT hepatic arteriography and CT during arterial portography (CTP) [2]. During hepatocarcinogenesis, most hepatocellular nodules show a deterioration of arterial blood flow before the loss of portal blood flow. Subsequently, as a result of a marked increase in neovascularized arteries, arterial blood flow becomes dominant. The portal blood flow decreases with the advancement of tumor, and the tumor is eventually fed mainly by arterial flow [3]. The changes of intranodular blood supply in a regenerative nodule, dysplastic nodule, and well-differentiated HCC have been well described [2 4]. dvanced HCC usually indicates moderately differentiated HCC or poorly differentiated HCC, and these two have been studied inseparably in the radiology literature until now [3, 4]. The hemodynamic changes in moderately differentiated HCC and poorly differentiated HCC have not been well evaluated radiologically or pathologically in a previous study, to our knowledge. The survival of patients with poorly differentiated HCC, however, is reportedly worse than that of those with moderately differentiated HCC in surgically resected cases [5]. In particular, Jonas et al. [6] revealed that the survival of patients with poorly differentiated HCC was significantly worse than survival of patients with well- and moderately differentiated HCC after liver transplantation. Therefore, it would be of clinical benefit if these two groups could be differentiated clinically or radiologically. We have previously reported W28 JR:190, January 2008

2 rterial lood Supply of HCC and Histologic Grading that some poorly differentiated HCCs showed a decreased arterial blood supply [7]. In terms of hepatocarcinogenesis, the number of unpaired arteries is substantially greater in HCCs than in cirrhotic nodules or dysplastic nodules and tends to correlate with the degree of arterial contrast enhancement [2, 3, 8]. The most widely used proliferation-associated marker is Ki-67. Ki-67 is a nuclear antigen present only in proliferating cells. detailed cell cycle analysis showed that the Ki-67 antigen is expressed in cells during the G 1, S, and G 1 M phases but not during the G 0 phase of the cell cycle [9, 10]. The diagnostic and prognostic value of Ki-67 immunostaining of human tumors has been well documented [11]. In HCC, Ki-67 expression was found to correlate closely with tumor growth rate [12] and was an independent prognostic indicator of patient disease-free and overall survival rates [13]. In this study, we hypothesized that arterial flow might, in general, decrease in the late stage of HCC development (moderately differentiated HCC to poorly differentiated HCC), whereas it increases in the early stage (welldifferentiated HCC to moderately differentiated HCC), as previously described [2 4]. The purpose of this study was to evaluate the hemodynamic patterns of HCCs on CT hepatic arteriography and CTP during the development of HCC, with a particular emphasis on its late stage, in correlation with the number of unpaired arteries and the expression of Ki-67 (proliferative activity index). Materials and Methods There were 199 consecutive HCC patients (212 nodules) who underwent surgical resection at our hospital from March 2003 to February Of these 199 patients, 26 patients (27 nodules) were excluded because they underwent preoperative radiofrequency ablation or transarterial chemotherapy. Of 173 patients (185 nodules), 136 patients (144 nodules) underwent CTP and CT hepatic arteriography preoperatively. Of these 144 nodules, 20, 92, and 32 nodules were proven to be well-differentiated HCC, moderately differentiated HCC, and poorly differentiated HCC, respectively, on the basis of the definitions provided by the Japanese Liver Cancer [14]. We randomly chose 20 of the 92 moderately differentiated HCCs and 20 of the 32 poorly differentiated HCCs. Finally, a total of 60 cases (20 well-differentiated HCCs, 20 moderately differentiated HCCs, and 20 poorly differentiated HCCs) comprised the study population. Tumor size was not taken into consideration for inclusion criteria. The mean size (± SD) of the well-differentiated HCCs, moderately differentiated HCCs, and poorly differentiated HCCs was 1.8 ± 0.6, 2.6 ± 1.5, and 4.2 ± 2.9 cm, respectively. The mean time interval between imaging (i.e., CT hepatic arteriography and CTP) and surgical resection was 14.2 ± 4.2 days. Our institutional review board does not require its approval or informed consent for the retrospective evaluation of patients records and images. CT Protocols CT hepatic arteriography and CTP were performed using a 4-MDCT unit (quilion, Toshiba) immediately after unenhanced scanning of the liver. The parameters for scanning were as follows: collimation, 3 mm; reconstruction, 5 mm; pitch, 5.5: amperage, 300 ms; kilovoltage, 120 kvp; and table speed, 16.5 mm per rotation. For CTP, data acquisition was started 30 seconds after the initiation of a transcatheter arterial injection of 100 ml of nonionic contrast material (iopamidol, 150 mg I/mL [Iopamiron 150, ayer HealthCare]) at 2.5 ml/s using an automated power injector. efore the contrast material was injected, a transarterial infusion of prostaglandin E 1 (10 mg) as a vasodilator was performed. Data acquisition of CT hepatic arteriography began 15 (first phase) and 30 (second phase) seconds after the initiation of a transcatheter hepatic arterial injection of ml of nonionic contrast material at a rate of ml/s using an automated power injector. The appropriate injection rate for CT hepatic arteriography was determined to be the maximum injection rate, which basically depended on the vessel caliber, that would not cause backward flow of contrast material on hepatic arteriography. The duration of the arterial injection was 20 seconds. Pathologic Evaluation For all 60 nodules, two experienced pathologists with 2 and 7 years of experience with liver pathology examined the resected specimens with no knowledge of the preoperative CT findings in a consensus fashion. Unpaired arteries in neoangiogenesis were not associated with the portal tract, fibrous tissue, septa, or other units (e.g., bile duct). The unpaired arteries were assessed according to established methods of vascular counting as follows [15]: the three neovascularized areas (hot spots) were originally identified at low magnification ( 40) and were circled in pen, then the absolute number of unpaired arteries was counted in a single field at the highest magnification ( 200) within every hot spot area. Immunohistochemistry Ki-67 was detected immunohistochemically by the streptavidin-biotin peroxidase method [16] using anti-ki-67 (MI-1, DakoCytomation). To measure the Ki-67 labeling index, more than 1,000 cells were counted in randomly selected high-power fields, and the number of Ki-67 positive nuclei was recorded in each field. The labeling index was defined as the ratio of Ki-67 labeled cells to the total number of cells counted and was expressed as percentages. ssessment Images of the largest axial sections of the tumor were evaluated by two radiologists with 11 and 19 years of experience with CT of the liver in a consensus fashion. ll interpretations were independently made from the axial CT images on a monitor (Coronis 3MP, arco), and the observers were completely blinded to the pathologic and clinical findings for each case. On the monitor, images obtained with routine window settings (window level, 100 H; window width, 500 H) were viewed. ll lesions were classified in one of the following groups on the basis of the enhancement patterns observed on CT hepatic arteriography and CTP: group 1, nodules that showed preserved portal perfusion on CTP and were hypo- or isoattenuating on CT hepatic arteriography (Fig. 1); group 2, portal perfusion defect on CTP and nodules were very hyperattenuating compared with noncancerous liver on CT hepatic arteriography (Fig. 2); group 3, portal perfusion defect on CTP and nodules were hyperattenuating compared with noncancerous liver on CT hepatic arteriography (Fig. 3); and group 4, portal perfusion defect on CTP and nodules were hypo- or isoattenuating compared with noncancerous liver on CT hepatic arteriography (Fig. 4). On CTP, preserved portal perfusion is defined as isoattenuation or decreased perfusion that is still partially preserved compared with noncancerous areas. s for the difference between groups 2 and 3, the lesions seen on CT hepatic arteriography in group 3 showed a hazy increase in the tumor attenuation level that is not associated with obscuration of the intratumoral vascular structure. In contrast, if a tumor showed obscuration of the vascular structure, the lesion was classified in group 2. In the case of a tumor showing various attenuation levels, the majority attenuation level was noted. ecause the intratumoral hypoattenuation area that exhibited no contrast enhancement in all phases was considered to be tumor necrosis, we excluded those areas from evaluation. We compared the attenuation pattern and the Ki-67 labeling index. We also compared the number of unpaired arteries and the Ki-67 labeling index. Statistical nalysis Continuous variables were expressed as means ± SD and tested using the Student s t test. The level of significance was set at a p value of less than 0.05 for all tests. Cramer s V with chi-square was used for radiologic pattern and tumor differentiation. The value of the level of association was interpreted as follows: JR:190, January 2008 W29

3 sayama et al. Fig year-old man with moderately differentiated hepatocellular carcinoma (group 1) in right lobe of liver. seconds after contrast material injection shows hypoattenuating mass (arrow) compared with noncancerous region. isoattenuating mass compared with noncancerous region. Number of unpaired arteries is one, and Ki-67 labeling index is 9. Fig year-old man with moderately differentiated hepatocellular carcinoma (group 2) in right lobe of liver. seconds after contrast material injection shows very hyperattenuating mass (arrow) compared with noncancerous region. perfusion defect compared with noncancerous region. Number of unpaired arteries is 13, and Ki-67 labeling index is 25. Fig year-old man with poorly differentiated hepatocellular carcinoma (group 3) in right lobe of liver. seconds after contrast material injection shows hazy hyperattenuating mass (arrows) compared with noncancerous region. perfusion defect compared with noncancerous region. Number of unpaired arteries is nine, and Ki-67 labeling index is , no relationship; , not generally useful; , weak; > , moderate; > , moderately strong; > , strong; > , very strong; > , worrisomely strong; > , redundant; and 1.00, perfect relationship. Spearman s rank correlation coefficient test was used to determine the correlation between the number of unpaired arteries and the Ki-67 labeling index. correlation coefficient value of up to 0.20 showed virtually no correlation; , weak correlation; , moderate correlation; , high correlation; and 0.81 or greater, very high correlation. W30 JR:190, January 2008

4 rterial lood Supply of HCC and Histologic Grading TLE 1: Radiographic Pattern and Pathologic Findings Results Details of the radiographic pattern and pathologic findings are shown in Table 1. The number of cases in groups 1, 2, 3, and 4 were 17 (all were well-differentiated HCCs), 14 (one well-differentiated HCC, 10 moderately differentiated HCCs, and three poorly differentiated HCCs), 23 (two well-differentiated HCCs, nine moderately differentiated HCCs, and 12 poorly differentiated HCCs), and six (one moderately differentiated HCC and five poorly differentiated HCCs), respectively (Cramer s V = , p < ). In hypervascular HCC (groups 2 and 3), three of 14 C Fig year-old man with poorly differentiated hepatocellular carcinoma (group 4) in right lobe of liver. seconds after contrast material injection from right hepatic artery shows hypoattenuating mass (arrows) compared with noncancerous region (arrowheads). perfusion defect (arrowheads) compared with noncancerous region. Number of unpaired arteries is three, and Ki-67 labeling index is 178. C, Immunohistochemical staining of Ki-67. Nuclei of tumor cells were diffusely positive for Ki-67 staining. Radiologic Pattern Well-Differentiated HCC Moderately Differentiated HCC Poorly Differentitated HCC Characteristic No. of cases Pathologic findings No. of unpaired arteries 2.1 ± ± ± ± ± ± ± 3.3 Ki-67 labeling index 0.7 ± ± ± ± ± ± ± 15.6 Note Dash ( ) indicates not applicable. Data are presented as mean ± SD or as value if there was only case. HCC = hepatocellular carcinoma. cases (21.4%) in group 2 were poorly differentiated HCCs, and 12 of 23 cases (52.2%) in group 3 were poorly differentiated HCCs. In group 4, five of six cases (83.3%) were poorly differentiated HCCs. The mean number of unpaired arteries in moderately differentiated HCC (10.5 ± 4.1) was significantly higher JR:190, January 2008 W31

5 rterial lood Supply of HCC and Histologic Grading Fig. 5 Relationship between CT during arterioportography and CT hepatic arteriography findings and Ki-67 labeling index. Note that Ki-67 labeling index of group 4 was significantly higher than those of other groups. Ki-67 Labeling Index p = p = p = p = p = p = than that in poorly differentiated HCC (4.7 ± 3.9, p < ) and in well-differentiated HCC (2.5 ± 1.5, p < ). The mean number of unpaired arteries in poorly differentiated HCC was significantly higher than that in well-differentiated HCC (p = ). In well-differentiated HCC, the highest Ki- 67 labeling index was observed in group 2 (3.0), followed by group 3 (2.6 ± 3.5) and group 1 (0.7 ± 0.6). In moderately differentiated HCC, the highest Ki-67 labeling index was observed in group 2 (10.6 ± 9.0), followed by group 4 (6.9) and group 3 (6.6 ± 6.7). In contrast, in poorly differentiated HCC, the highest Ki-67 labeling index was observed in group 4 (40.7 ± 15.6), followed by group 3 (26.1 ± 17.1) and group 2 (9.1 ± 7.8). The number of unpaired arteries showed a strong correlation with Ki-67 labeling index in welldifferentiated HCC and moderately differentiated HCC (rho = 0.673, p < ). In contrast, the number of unpaired arteries showed a moderate inverse correlation with Ki-67 labeling index in poorly differentiated HCC (rho = 0.540, p = ). The relationship between the radiologic findings and the Ki-67 labeling index of all HCCs is shown in Figure 5. The Ki-67 labeling indexes of group 2 (9.8 ± 8.3), group 3 (16.4 ± 16.4), and group 4 (35.1 ± 19.6) were higher than that of group 1 (7.1 ± 5.5) (p = , p = , and p < , respectively). The Ki-67 labeling index of group 4 was higher than those of group 2 and group 3 (p = and , respectively). The Ki-67 labeling index of group 3 tended to be higher than that of group 2, but the difference was not significant (p = 0.167). Discussion Investigators have reported a correlation between the blood supply and the grade of malignancy of hepatocellular nodules in radiologic and pathologic analyses [2 4]. In these articles, researchers focused on precancerous lesion and early-stage HCCs namely, low-grade and high-grade dysplastic nodules and well-differentiated HCC. However, few previous reports have discussed the vascular changes that occur in the late stage of advancement and the direct relationship between the arterial blood flow of the tumor and the tumor s proliferative activity. We have not been able to forecast the proliferative activity of the advanced HCC solely on the basis of radiologic findings. s shown in Table 1, well-differentiated HCCs mainly belonged to group 1, moderately differentiated HCCs mainly belonged to group 2 followed by group 3, and poorly differentiated HCCs mainly belonged to group 3 followed by group 4. The number of unpaired arteries increased as the histologic grade progressed from well-differentiated HCC to moderately differentiated HCC, but it decreased as the tumor grade progressed from moderately differentiated HCC to poorly differentiated HCC. These results suggest that the vascularity of poorly differentiated HCC tends to decrease in comparison with moder- ately differentiated HCC both radiologically and pathologically. In the current study, regarding hypervascular advanced HCC, the Ki-67 labeling index of group 2 (10.6 ± 9.0) was higher than that of group 3 (6.6 ± 6.7) in moderately differentiated HCC, but the Ki-67 labeling index of group 2 (9.1 ± 7.8) was lower than that of group 3 (26.1 ± 17.1) in poorly differentiated HCC (the difference was not significant). Furthermore, the Ki-67 labeling index of group 4 in poorly differentiated HCC was the highest of all the groups. The number of unpaired arteries of well-differentiated HCC and moderately differentiated HCC and of poorly differentiated HCC showed a strong correlation and a moderate inverse correlation with Ki-67 labeling index, respectively. These results also suggest that arterial blood supply increases in the early stage of advancement but that, conversely, it decreases in the late stage of advancement. ccording to this concept, both groups 3 and 4 may theoretically consist of two different subgroups: one with moderate tumor aggressiveness exhibiting an intermediate Ki-67 labeling index and the other with high tumor aggressiveness exhibiting a high Ki-67 labeling index. schematic presentation of this concept regarding the sequential changes of intratumoral arterial blood supply is summarized in Figure 6. The numbers of HCCs belonging to groups 3 and 4 and showing intermediate Ki-67 labeling index (here, it was defined as < 10) that is, those considered to be on the left side of the peak (group 3' and group 4') were 10 (eight moderately differentiated HCCs and two poorly differentiated HCCs) and one (moderately differentiated HCC), respectively. Two cases of well-differentiated HCC also belonged to group 3'. The numbers of HCCs belonging to group 3 and group 4 and showing high Ki-67 labeling index ( 10) that is, those considered to be on the right side of the peak (group 3 and group 4) were 11 (one moderately differentiated HCC and 10 poorly differentiated HCCs) and five (all were poorly differentiated HCCs), respectively. Thus, the HCCs with the group 3' or group 4' hemodynamic pattern and intermediate Ki-67 labeling index were well-differentiated HCC (n = 2) or moderately differentiated HCC (n = 9) (total = 11) rather than poorly differentiated HCC (n =2) and those with the group 3 or group 4 hemodynamic pattern and high Ki-67 labeling index were poorly differentiated HCC (n = 15) rather than moderately differentiated HCC (n =1) (p = , Fisher s exact test). Figure 5 shows that the correlation between the Ki-67 labeling index and the hemodynamic sequence order of groups 1, 2, 3, and JR:190, January 2008 W32

6 rterial lood Supply of HCC and Histologic Grading Neovascularized rterial Flow Ki-67 Labeling Index Equal to noncancerous liver Well- Differentiated HCC Moderately Differentiated HCC dvancement of Pathological Grading 1 1 4' 4' 3' 2 3' Proliferative activity was significant (rho = 0.677, p , Spearman s rank correlation); however, when the sequence was changed to groups 1, 4', 3', 2, 3, and 4, the correlation became stronger (rho = 0.872, p < ) (Fig. 7). This finding may support our hypothetical concept shown in Figure 6. lthough strict differentiation between these two subgroups of group 3 and group 4 namely, group 3' versus group 3 or group 4' versus group 4, in other words, moderately differentiated HCC versus poorly differentiated HCC solely by the presented radiologic method (CT hepatic arteriography and CTP) may be difficult, we believe these imaging techniques can provide important information in an approximate estimation of the proliferative activity of the tumor (Fig. 5). new radiologic approach, such as diffusionweighted imaging or calculated apparent diffusion coefficient values on MRI [17], may be Poorly Differentiated HCC helpful in this strict differentiation of group 3' versus group 3 or of group 4' versus group 4. From the standpoint of implications for patients, Jonas et al. [6] reported that vascular invasion and histopathologic grade of differentiation were identified as the only prognostic parameters in liver transplant recipients. Thus, in planning liver transplantation, evaluation of arterial blood flow can contribute to predict a worse prognosis in patients with hypovascular advanced HCC. Furthermore, transarterial infusion chemotherapy may be not effective for hypovascular advanced HCCs compared with hypervascular HCCs. We should choose alternative treatments as soon as possible because such tumors will show rapid progression due to the high proliferative activity of the tumor cells. In malignant neoplasms, angiogenesis is also required for tumor growth, progression, and metastasis [18 20]. The supply of oxygen Fig. 6 Diagram shows changes of intratumoral arterial blood supply. In welldifferentiated hepatocellular carcinoma (HCC) and moderately differentiated HCC, arterial blood supply increases as histologic grade progresses. Consequently, arterial blood supply decreases in poorly differentiated HCC as tumor grade advances. HCCs belonging to group 3 and group 4 and showing intermediate Ki-67 labeling index (here, it was defined as < 10) that is, those considered to be on left side of peak (group 3' and group 4') mainly consisted of moderately differentiated HCCs. HCCs belonging to group 3 and group 4 and showing high Ki-67 labeling index ( 10) that is, those considered to be on right side of peak (group 3 and group 4) mainly consisted of poorly differentiated HCCs. (Strictly speaking, it seems to be incompatible to place poorly differentiated HCC showing group 3' or group 4' into a portion labeled as well-differentiated HCC or moderately differentiated HCC. However, such tumor is rare.) Moderately differentiated HCC on line shows same degree of arterial blood flow as poorly differentiated HCC on line, but proliferative activity of HCC on line is higher than that of HCC on line. (It is difficult to distinguish from solely by means of CT hepatic arteriography and CT during arterioportography.) Poorly differentiated HCC on line shows hypovascular and very high proliferative activity. It is possible to point out poorly differentiated HCC on line, and thus we can predict proliferative activity of this type of tumor. Fig. 7 Relationship between CT during arterioportography and CT hepatic arteriography findings and Ki-67. In this figure, group 3 was divided into group 3 and group 3', and group 4 was divided into group 4 and group 4'. Correlation between Ki- 67 labeling index and hemodynamic sequence order of group 1, 4', 3', 2, 3, and 4 (rho = 0.872) was stronger than that between Ki-67 labeling index and sequence order of group 1, 2, 3, and 4 (rho = 0.677). and nutrition from the tumor vessels is believed to be essential for the growth of solid tumors [21]. During multistep hepatocarcinogenesis, the arterial blood flow increases from hypovascular well-differentiated HCC to hypervascular classical HCC [2 4]. During this process, the proliferative activity of the tumor cells also increases. In contrast, the Ki-67 labeling index showed a moderate inverse correlation with arterial blood flow in poorly differentiated HCC. Mineura et al. [21] previously reported that high-grade gliomas tended to show lower oxygen consumption than lower-grade gliomas using 15 O PET, and they speculated that high-grade gliomas may have anaerobic metabolism in contrast to the aerobic metabolism of low-grade tumors [21]. Considering our results, a similar mechanism may apply to HCC. Yasuda et al. [22] showed that glycolysis induced by hypoxia JR:190, January 2008 W33

7 sayama et al. inducible factor 1 (HIF-1) is the predominant energy source under the hypoxic environment in metastatic liver cancers, and some clinicians have pretreated HCC by transarterial embolization. It may be presumed that in some poorly differentiated HCCs, tumor cells acquire a metabolic profile of increased glycolysis through the HIF-1 regulated pathway, thus enabling them to proliferate more rapidly under hypoxia. In other words, aerobic metabolizing as the energy source in hypervascular HCC may switch to glycolysis in some poorly differentiated HCCs that show less arterial blood flow. The present study has some limitations. First, there is a possibility that the number of unpaired arteries does not directly reflect the attenuation level of HCC on CT hepatic arteriography. The degree of contrast enhancement of the nodular HCC in the arterial phase tended to correlate with the number of unpaired arteries [8]; however, in our recent study [7], the number of unpaired arteries showed weak correlation with the attenuation level on CT hepatic arteriography and it has been suggested that in addition to arterial blood flow, microvascular permeability is important in the contrast enhancement of a lesion [8, 23]. Second, we did not use a quantitative measure of the tumor on CT hepatic arteriography. However, individual differences in the degree of liver fibrosis can greatly affect the attenuation of the liver tumor and liver parenchyma. This difficulty in evaluating these hepatocellular lesions on the basis of blood supply may add limitations to this study [2]. In conclusion, we showed that arterial blood flow decreased in the late stage of HCC development both radiologically and pathologically. lthough exact differentiation between poorly differentiated HCC and moderately differentiated HCC based on CTP and CT hepatic arteriography is still limited, hypovascular advanced HCC showed higher proliferative activity than hypervascular advanced HCC. cknowledgments We thank Yoshihiko Maehara, Professor and Chair, Department of Surgery and Science, Kyushu University, for providing clinical data and Tsuneyoshi Masazumi, Professor and Chair, Department of natomic Pathology, Kyushu University, for providing pathologic data. We also thank Tsuyoshi Tajima, kihiro Nishie, Masakazu Hirakawa, Kousei Ishigami, and Daisuke Kakihiara, Department of Clinical Radiology, Kyushu University, for editing this manuscript. References 1. Okuda K. Hepatocellular carcinoma: clinicopathological aspects. 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