Immunohistochemical demonstration of sinusoidal capillarization in human benign liver tumors: distinction from neoangiogenesis

Hepatology Research 28 (2004) 87–93

Immunohistochemical demonstration of sinusoidal capillarization in human benign liver tumors: distinction from neoangiogenesis Kazuhiro Hirohashi a , Takatsugu Yamamoto a,∗ , Shogo Tanaka a , Taichi Shuto a , Shoji Kubo a , Hiromu Tanaka a , Akishige Kanazawa a , Takahiro Uenishi a , Masao Ogawa a , Katsu Sakabe a , Tsuyoshi Ichikawa a , Takashi Ikebe a , Kenji Kaneda b , Masami Sakurai c , Hiroaki Kinoshita a a

Hepato-Biliary-Pancreatic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan b Senior Health Facility Bellflower, Osaka, Japan c Department of Food and Nutritional Science, Kobe Women’s Junior College, Kobe, Japan Received 5 November 2002; received in revised form 12 August 2003; accepted 23 October 2003

Abstract To better understand how benign hepatocellular tumors acquire nutrient vessels, we investigated the angioarchitecture of the smallest vessels in hepatocellular adenoma (HA) and focal nodular hyperplasia (FNH). In biopsy or resection specimens, microscopic angioarchitecture was characterized in terms of intratumoral fibrous septa and portal tract. Immunohistochemistry for CD34, factor-VIII-related antigen (F-VIII), type IV collagen (collagen IV), ␣-smooth muscle actin (␣-SMA), and CD68 was performed. HA had no portal tract in fibrous septa although FNH showed portal tract in fibrous septa. The smallest vessels in HA stained diffusely for CD34, and arterial segments of the smallest vessels were stained for F-VIII, and collagen IV. Pericyte surrounding the smallest vessels in HA were stained for ␣-SMA. The smallest vessels in all but one FNH specimen stained for CD34, F-VIII, collagen IV, and ␣-SMA along portal tract or the artery. Both HA and FNH stained for CD68, as did normal liver. The results indicate that the smallest vessels in HA and FNH are incomplete capillaries, that have both characters of sinusoid and capillaries morphologically and phenotypically, i.e., capillarized sinusoid as in chronic hepatitis or early well-differentiated hepatocellular carcinoma. Benign hepatocellular tumors such as HA and FNH would acquire new vessels gradually by sinusoidal capillarization, not by rapid arterial angiogenesis. © 2003 Published by Elsevier B.V. Keywords: Hepatocellular adenoma; Focal nodular hyperplasia; CD34; Factor VIII-related antigen; Sinusoidal endothelium; Capillary; Sinusoidal capillarization

1. Introduction Hepatic tumors, especially hepatocellular tumors, often show arterial hyperdynamic flow on imaging. These neoplasms are fed mainly by arterial flow in contrast to normal liver fed by both portal and arterial flow. Hepatocellular carcinoma (HCC), a typical malignant hepatocellular neoplasm, is well known to be fed by arterial flow through capillaries, not by venous flow through sinusoids. Many investigators have reported that the smallest vessels of advanced HCC are Abbreviations: HA, hepatocellular adenoma; FNH, focal nodular hyperplasia; F-VIII, Factor VIII-related antigen ∗ Corresponding author. Tel.: +81-6-6645-3841; fax: +81-6-6646-6057. E-mail address: [email protected] (T. Yamamoto). 1386-6346/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.hepres.2003.10.003

capillaries, and capillaries in advanced HCC are immunohistochemically stained with CD34, factor VIII-related antigen, and Ulex Europaeus-1 lectin, which usually do not stain the endothelium of liver sinusoids [1–3]. However, some small well-differentiated HCCs are surrounded by pre-existing sinusoids not withstanding the tumor’s arterial blood supply [4]. While the lesions of hepatocellular adenoma (HA), benign hepatocellular neoplasm, and focal nodular hyperplasia (FNH) representing hepatocytic hyperplasia, are fed by arterial flow [5], the nature of the smallest vessels encircling their parenchyma remains unclear. We immunohistochemically investigated the endothelium of the smallest vessels and interstitial cells like Kupffer cell and fat-storing stellate cell at the periphery of the smallest vessels in these benign hepatocellular tumors to determine the process of vascularization.

88

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

2. Materials and methods We encountered two cases of HA and six of FNH between 1988 and 2001. Seven patients had no history of hepatitis, use of hormonal drugs, or metabolic disease; one patient (case 6) had a history of pancreatectomy for islet cell tumor. Clinically, all patients except case 2 had no history of liver dysfunction. Arterial angiography definitively revealed findings typical for FNH in two cases. No diagnostic biopsy or surgical treatment was performed in these two patients. The two patients with HA and two other FNH patients were treated with surgical resection (cases 1, 2, 5, and 6). The two remaining patients with FNH were diagnosed by needle biopsy, eliminating the need for surgical treatment (cases 3 and 4). The two HA cases (cases 1 and 2) and two FNH cases (cases 5 and 6) were not diagnosed until postoperative microscopic examination. All six patients gave consent to the study, and we examined the six specimens as well as clinical data including imaging findings. Resection and biopsy specimens were fixed in 10% formalin, embedded in paraffin, cut at a thickness of 3 ␮m, and stained with hematoxylin and eosin for histologic diagnosis. Intratumoral fibrous septa, portal triads, isolated arteries, and veins were examined microscopically. Immunohistochemistry was performed using the same formalin-fixed and paraffin-embedded tissue blocks using components from the Envision-plus detection system

(DAKO, Kyoto) and antibodies against CD34 (monoclonal; DAKO, Kyoto), factor VIII-related antigen (F-VIII, monoclonal; DAKO, Kyoto), type IV collagen (collagen IV, monoclonal; DAKO, Kyoto), ␣-smooth muscle actin (␣-SMA, monoclonal; DAKO, Kyoto), or CD68 (monoclonal; DAKO, Kyoto). Two no tumor liver specimens obtained at resection of colon cancer, metastatic to the liver were used as control. Under a 40× objective, cells stained with CD68 were counted in four fields (total, 0.05 mm2 ), and the mean was calculated. CD34 is an intercellular adhesion protein, and F-VIII is a product of endothelium. Their expressions indicate either ordinary endothelium or capillarized endothelium of sinusoids. Collagen IV is one type of collagen fiber; its expression indicates the presence of a basement membrane, an intercellular protein that separates parenchymal cells from ordinary endothelium but not sinusoids [3]. ␣-SMA is a fiber present in fibroblasts, activated stellate cells like myofibroblast, smooth muscle, and pericytes [6]. CD68 is a surface antigen present on macrophages and Kupffer cells [7]. 3. Results 3.1. Macroscopic findings All HA had distinct margins, and were encapsulated by thin fibrous tissue. HA showed a bulging cut surface, a light tan color, focal hemorrhage, and absence of fibrous septa.

Fig. 1. Histopathologic feature of HA. The parenchymal cells of HA show few atypia, and form lines resembling hepatic cords (20×, hematoxylin and eosin stain). There is arteriole isolated and no portal tract. Inset demonstrates arteriole without cholangiolar ductule or portal vein (100×, hematoxylin and eosin stain).

Table 1 Profiles and histopathologic characteristics of HA and FNH cases Gender Age Tumor size (cm)

Histologic diagnosis

Preoperative diagnosis

Chief complaint Resection/ biopsy

Presence of Glisson’s sheath

CD34

F-VIII

Collagen IV

␣-SMA

CD68 positive cells (mean)

1

M

19

HA

HCC

Epigastralgia

Resection

Artery only

M

36

10

HA

HCC

Resection

Artery only

3

M

14

5

FNH

FNH

Liver dysfunction Epigastralgia

Biopsy

Present

4

M

25

9

FNH

FNH

Palpable mass

Biopsy

Present

5

F

31

3.8

FNH

HA

Epigastralgia

Resection

Present

6

F

48

1.7

FNH

Metastsis of islet cell tumor of pancreas

Incidentally

Resection

Present

SVs beside artery were stained SVs beside artery were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained

SVs beside artery were stained SVs beside artery were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained

SVs beside artery were stained SVs beside artery were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs were stained diffusely

62

2

SVs were stained diffusely SVs were stained diffusely SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs beside Glisson’s sheath were stained SVs were stained diffusely

5.3

45 39

61

57

44

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

Case no.

HA, hepatocellular adenoma; FNH, focal nodular hyperplasia; HCC, hepatocellular carcinoma; F-VIII, factor VIII-related antigen; Collagen IV, type IV collagen; ␣-SMA, ␣-smooth muscle actin; SVs, smallest vessels.

89

90

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

Fig. 2. Histopathologic feature of FNH. The parenchymal cells of FNH show few atypia, and form lines resembling hepatic cords (20×, hematoxylin and eosin stain). There is degenerated portal tract. Inset demonstrates artery hypertrophying and hyperplastic cholangiolar ductules in the portal tract (100×, hematoxylin and eosin stain).

FNH resected surgically showed a distinct margin but no fibrous capsule. These lesions bulged slightly from the cut surface and were light tan, showing no hemorrhage. The tumor of case 5 had fibrous septa, but no central scar formation; the tumor in case 6 had no fibrous septa or central scar. In cases 1, 2, 5, and 6, there was no significant change in non-tumor region of the liver macroscopically. 3.2. Microscopic findings HA had a hemorrhagic area within the tumor and showed thick-walled arterioles and veins suggestive of draining vessels. The parenchymal cells of HA were slightly hypercellularity, but no cellular atypia or abnormality of nuclear:cytoplasmic ratio was seen. These tumors showed the smallest vessels within parenchymal cords like sinusoids. In some places, the smallest vessels were dilated, resembling those in peliosis hepatis. No triad in portal tract was present (Fig. 1). One resected FNH lesion had portal tract and fibrous septa that contained thick-walled arteries and hyperplastic cholangiolar ductules. No central fibrous scar was present. The other resected FNH had portal tract and no fibrous septa. Specimens from both biopsied FNH lesions had portal tract, which consisted of hyperplastic bile ductules and no fibrous septa. No parenchymal cells in these four FNH specimens showed hypercellularity, cellular atypia, or abnormal nuclear:cytoplasmic ratios (Fig. 2).

In cases 1, 2, 5, and 6, there was no significant change in non-tumor region of the liver microscopically. 3.3. Immunohistochemical findings Results of immunohistochemical staining for CD34, F-VIII, collagen IV, ␣-SMA, and CD68 are summarized in Table 1. The smallest vessels of HA were stained for CD34 diffusely, and the smallest vessels stained for F-VIII, collagen IV, and ␣-SMA were seen near wall-thick arteries in patches (Fig. 3A–C). Capillaries in HA had many macrophages along their sides, mimicking Kupffer cells in liver sinusoids (Fig. 3D). Additionally, three of four FNH lesions were stained for CD34, F-VIII, collagen IV, and ␣-SMA along the fibrous septum (Fig. 4A-C). In case 6, CD34 and ␣-SMA diffusely stained the smallest vessels. CD68 stained all FNH lesions in a diffuse manner (Fig. 4D). No significant differences in numbers of CD68-positive cells were seen between HA, FNH, and control liver tissue.

4. Discussion Most hepatocellular tumors show arterial hyperdynamic flow on imaging, and receive mainly arterial flow. Normally, the liver receives flow from both portal veins and hepatic arteries.

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

91

Fig. 3. Histopathologic feature of HA. The smallest vessels near arterioles are stained for F-VIII (A). The basement membrane of smallest vessels near arterioles are scatteringly stained for collagen IV (black arrows) (B). The pericytes of smallest vessels near arterioles mimicking hepatic stellate cell are locally stained for ␣-SMA (black arrows)(C). Intraluminal macrophages stained for CD68 (D) diffusely, mimicking Kupffer cells.

Hepatocellular carcinoma (HCC), a typical malignant hepatocellular neoplasm, is supplied by tumor arteries and capillaries, not by the sinusoids of the portal systems. Many investigators reported that capillaries in HCC were immunostained for CD34, factor VIII-related antigen, and Ulex Europaeus-1 lectin. Many authors have hypothesized that capillaries in HCC represent neoangiogenesis initiated by malignant parenchymal cells [1–3]. Other hepatocellular tumors including HA, a benign hepatocellular neoplasm, and FNH, an area of hepatocytic hyperplasia also are fed by arterial flow. Many reports have indicated that HA have fibrous capsule, tumor arteries isolated from triad in portal tract, and no portal tract. On angiography, these isolated arteries enter the tumor to ramify extensively [8,9]. In contrast, FNH has no fibrous capsule and has a central scar or fibrous septum which many investigators consider to have been derived from portal tract because of the presence of portal triad in the fibrous septum. Outlying parenchymal cells of FNH adjoin normal hepatocytes, and the smallest vessels arise from normal sinusoid at the periphery. Numerous researchers have investigated the micro-angioarchitecture of HCC [8,10]. Although many studies have considered blood flow in FNH and HA, little is known about their micro-angioarchitecture. The present immunohistochemical study revealed an incomplete distribution of basement membranes of the

smallest vessel, and the diffuse presence of peri- or intra-microvascular resident macrophages in both HA and FNH. A capillary phenotype was expressed in the smallest vessels close by thick-walled arteries in HA, and portal tract in FNH. On the other hand, our previous study demonstrated that the smallest vessels of the small HCCs encapsulated that had demonstrated arterial hyperdynamic flow on angiography, were diffusely stained for F-VIII, collagen IV immunohistochemically [3]. Contrary to our expectation, the results indicate that both HA and FNH were fed by atypical smallest vessels with some features of sinusoids. In one process of capillary acquisition, neoangiogenesis as seen in advanced HCC or hepatoblastoma, malignant cells produce angiogenetic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor (TGF), that induce budding of capillaries from arteries [11–13]. Additionally, rapid growth of the tumor compresses the surrounding liver and portal tract. Fibrous interstitial tissue is produced, and with this interstitium arterioles enter into the tumor. A different process of acquisition of capillaries, sinusoidal capillarization, is seen in chronic hepatitis such as chronic viral hepatitis, or lupoid hepatitis [14]. In this sequence, continuous slow destruction by inflammatory cells activates stellate cells and interstitial tissue adjoin portal tract to produce collagen fibers, actin filaments, and

92

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

Fig. 4. Histopathologic feature of FNH. The smallest vessels near degenerating portal tract (white arrows) are stained for CD34 (A). The basement membrane of smallest vessels near degenerating portal tract are locally stained for collagen IV (black arrows) (B). The pericytes of smallest vessels near arterioles mimicking hepatic stellate cell are locally stained for ␣-SMA (black arrows) (C). Intraluminal macrophages stained for CD68 (D) diffusely, mimicking Kupffer cells.

certain chemical mediators. We believe that this process of fibrosis gradually shifts the main blood flow from portal to arterial, and that the inflammatory effect and arterial flow incompletely change sinusoidal endothelium to capillaries [10,15]. The present result in HA and FNH indicates that both tumors are fed by arterial flow through such capillarized sinusoids. The blood supply of FNH consisted of mainly arterial flow with a smaller amount of portal flow. These alterations of arterial and portal flow resemble the blockade of portal flow by periportal inflammation that is seen in viral hepatitis. Some investigators suggest that increased blood pressure in sinusoids, high (arterial) oxygenation levels, or TGF stimulates activity of endothelium leading to morphologically evident capillarization [1,7,10]. The present results in FNH are consistent with the process of sinusoidal capillarization. A forced analogy between HCC and HA focusing on the presence of a fibrous capsule and tumor arteries has led to a belief that capillaries in HA arise from neoangiogenesis. The present results, however, demonstrated altered sinusoids, not neoangiogenetic capillaries. The sequential morphologic changes of sinusoidal capillarization have been reported in rat hepatic neoplasms, and fenestrated endothelium, intraluminal Kupffer cells, and

stellate cell with lipid droplets may be found even in some human HCC [3,16,17]. Sinusoidal capillarization appear to be the node of acquisition of blood supply in HA. Some investigators [4,18,19] reported direct anastomosis between sinusoid and arterioles (arterio-sinus twig) not only in the portal tract but also in the hepatic lobule. In the HA, a chemical mediator might selectively induce hypertrophy in the twig, and the tumor would compress portal tract to form a capsule. In conclusion, the present study indicated the presence of sinusoidal capillarization as opposed to neoangiogenesis in HA and FNH. The presence of sinusoidal capillarization in HA and FNH suggests that ferumoxides-enhanced magnetic resonance and immunohistochemistry the discriminate sinusoidal from capillary cells are useful in the differential diagnosis of HA from advanced HCC, when a tumor with hyperdynamic arterial flow on angiography or dynamic computed tomography is found. While, the arterial hyperdynamic flow in HA and FNH suggests that dynamic imaging series such as angiography, and dynamic computed tomography are useful in the differential diagnosis of HA from early stage of well-differentiated HCC because most of the early HCCs do not demonstrate arterial hyperdynamic flow on dynamic imaging series.

K. Hirohashi et al. / Hepatology Research 28 (2004) 87–93

References [1] Kong CS, Appenzeller M, Ferrel LD. Utility of CD34 reactivity in evaluating focal nodular hepatocellular lesion sampled by fine needle aspiration biopsy. Acta Cytologica 2000;44:218–22. [2] Ruck P, Xiao JC, Kaiserling E. Immunoreactivity of sinusoids in hepatoblastoma: an immunohistochemical study using lectin Ulex Europaeus-1 lectin and antibodies against endothelium-associated antigens, including CD34. Histopathology 1995;26:451–5. [3] Yamamoto T, Ikebe T, Mikami S, Shuto T, Hirohashi K, Kinoshita H, et al. Immunohistochemistry and angiography in adenomatous hyperplasias and small hepatocellular carcinomas. Pathol Int 1996;46:364– 71. [4] McCuskey R. Hepatic microcirculation. In: Bioulac-Sage P, Balabaud C, editors. Sinusoids in human liver: health and disease. Rijswijk: The Kupffer Cell Foundation; 1988. p. 151–64. [5] Welch TJ, Sheedy II PF, Johnson CM, Stephens DH, Charboneau JW, Brown MJ, et al. Focal nodular hyperplasia and hepatic adenoma: comparison of angiography, CT, US, and scintigraphy. Radiology 1985;156:593–5. [6] Enzan H, Himeno H, Iwamura S, Onishi S, Saibara T, Yamamoto Y, et al. Alpha-smooth muscle actin-positive perisinusoidal stromal cells in human hepatocellular carcinoma. Hepatology 1994;19:895– 903. [7] Tanaka M, Nakashima O, Wada Y, Kage M, Kojiro M. Pathomorphological study of Kupffer cells in hepatocellular carcinoma and hyperplastic nodular lesions in the liver. Hepatology 1996;24:807–12. [8] Hoe LV, Baert AL, Gryspeerdt S, Vandenbosh G, Nevens F, Steenbergen WV, et al. Dual-phase helical CT of the liver: value of an early-phase acquisition in the differential diagnosis of noncystic focal lesions. Am J Roentgenol 1997;168:1185–92. [9] Grazioli L, Federle MP, Ichikawa T, Balzano E, Nalesnik M, Madariaga J. Liver adenomatosis: clinical, histopathologic, and imaging findings in 15 patients. Radiology 2000;216:395–402.

93

[10] Kondo F, Nagao T, Sato T, Tomizawa M, Kondo Y, Matsuzaki O, et al. Etiological analysis of focal nodular hyperplasia of the liver, with emphasis on similar tumor vasculatures to nodular regenerative hyperplasia and idiopathic portal hypertension. Pathol Res Pract 1998;194:487–95. [11] Yamaguchi R, Yano H, Iemura A, Ogasawara S, Haramaki M, Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology 1998;28:68–77. [12] Yoshiji H, Kuriyama S, Yoshii J, Yamazaki M, Kikukawa M, Tsujinoue H, et al. Vascular endothelial growth factor tightly regulates in vivo development of murine hepatocellular carcinoma cells. Hepatology 1998;28:1489–96. [13] Motoo Y, Sawabu N, Yamaguchi Y, Terada T, Nakanuma Y. Sinusoidal capillarization of human hepatocellular carcinoma: Possible promotion by fibroblast growth factor. Oncology 1993;50:270–4. [14] Schaffner F, Popper H. Capillarization of hepatic sinusoids in human. Gastroenterol 1963;44:239–42. [15] Fukukura Y, Nakashima O, Kusaba A, Kage M, Kojiro M. Angioarchitecture and blood circulation in focal nodular hyperplasia. J Hepatol 1998;29:470–5. [16] Yamamoto T, Kaneda K, Hirohashi K, Kinoshita H, Sakurai M. Sinusoidal capillarization and arterial blood supply continuously proceed with the advance of the stages of hepatocarcinogenesis in the rat. Jpn J Cancer Res 1996;87:442–50. [17] Yamamoto T, Hirohashi K, Kaneda K, Ikebe T, Mikami S, Uenishi T, et al. Relationship of microvascular type to tumor size, arterialization and dedifferentiation in human hepatocellular carcinoma. Jpn J Cancer Res 2001;92:1207–13. [18] Yamamoto K, Sherman I, Phillips MJ, Fisher MM. Three-dimensional observations of the hepatic arterial terminations in rat, hamster and human liver by scanning electron microscopy of microvascular casts. Hepatology 1985;5:452–6. [19] Tajiri S. The terminal distribution on the hepatic artery. Acta Med Okayama 1960;14:215–25.