Hepatocellular carcinoma (HCC) is the fifth most common

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1 Vascular Invasion and Herniation by Hepatocellular Carcinoma in Cirrhosis A Wolf in Sheep s Clothing? Alberto Quaglia, MD, PhD, MRCPath; Nazanin Etessami, MD; Rosalind Sim, FIBMS; John Difford, FIBMS; A. P. Dhillon, MD, FRCP, FRCPath Context. Vascular invasion is an important diagnostic and prognostic feature of hepatocellular carcinoma (HCC) in cirrhosis. Intravascular free-floating tumor clusters (IvCs) of HCC are found histologically in the vicinity of HCC. Thrombus formation is not seen morphologically in association with these IvCs, which are usually covered by endothelium. Objective. Our hypothesis is that these IvCs are the result of a nondestructive form of vascular invasion by HCC, and we tried to define this aspect of microvascular invasion more accurately. Design. Tissue sections were stained with hematoxylineosin, and consecutive sections were stained for fibrin (Martius scarlet blue, fibrinogen), platelets (factor XIIIa), smooth muscle actin, and endothelium (CD34). We studied cirrhotic livers removed at transplantation between 1997 and Of the livers studied, 35 of 81 consecutive cirrhotic livers contained HCC, and 17 showed microscopic vascular invasion. Five of these 17 cases showed IvCs and were subjected to the study. Main Outcome Measure. Presence or absence of thrombus formation in association with IvC. Results. Usually, IvCs were covered by endothelium, and no associated thrombus formation was seen. In 1 case of HCC, thrombus formation was seen focally in association with disruption of the endothelial coating. Conclusions. We propose that the endothelial-lined trabecular structure of HCC everts, frondlike, via vascular structures within the tumor capsule into peritumoral vascular lumens without destruction of the endothelial coating. This may protect these HCC tumor projections from thrombus formation but may also act as a barrier to tumor extravasation, and this may be exploited from a therapeutic point of view. (Arch Pathol Lab Med. 2005;129: ) Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. 1 It usually arises in cirrhotic livers. Liver transplantation remains the most effective treatment option in selected patients. Recurrence of HCC after transplantation affects a variable proportion of patients, with recurrence rates of up to 25% in patients with early HCC. 2 Various clinicopathologic variables have been described as predictive of posttransplant recurrence. Vascular invasion is one of these factors, being associated with tumor recurrence and survival. 3 5 Vascular invasion can be identified at the radiologic or pathologic level. At the radiologic level, vascular invasion usually means infiltration of the portal vein or its main branches. At a pathologic level, vascular invasion may be evident during gross examination of resection or explant specimens (although macroscopic portal vein involvement may be visible on pretreatment imaging, representing an exclusion criterion for surgery), or it may be identified histologically. According to general principles of cancer biology, vascular invasion is a characteristic of neoplasms at a stage of tumor progression when tumor cells have developed a sufficiently evolved phenotype to invade blood vessels and Accepted for publication January 11, From the Department of Histopathology, Royal Free Hospital, London, United Kingdom. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Alberto Quaglia, MD, PhD, MRCPath, Department of Histopathology, Royal Free Hospital, Pond Street, London NW3 2QG, United Kingdom ( alberto.quaglia@royalfree.nhs.uk). potentially begin the distant metastatic process. 6 For tumors of epithelial origin, stromal invasion is a consequence of a complex evolutionary process that includes loss of intercellular adhesion, detachment from the basal membrane, digestion of extracellular matrix, and migration. Stromal invasion includes invasion of the wall of vascular structures, rupture of the endothelium, and penetration into the vascular lumen with access to blood and lymphatic flow. After detachment, a proportion of circulating tumor cells that have the capability of surviving in the blood circulation may adhere to or wedge into vessels at a distant site, egress, and establish a distant metastasis. According to this model, vascular invasion is a destructive process and should therefore be associated at the histologic level with signs of vascular destruction. In other words, at sites of vascular invasion one would expect to see disruption of the vascular wall, rupture of the endothelium, and associated thrombus formation. In HCCs in cirrhotic livers removed at transplantation, the presence of tumor clusters floating within the lumen of vascular spaces at the periphery of tumor nodules may be seen even in the absence of destructive infiltration of adjacent stroma. This appearance may be difficult to distinguish from artifactual spillage of friable tumor material into vessel spaces. This microvascular invasion pattern of HCC appears to represent a process of permeation rather than stromal destruction. In particular, thrombus formation in association with these tumor clusters may be absent. In our laboratory, we carry out immunostaining for CD34, among Arch Pathol Lab Med Vol 129, May 2005 Vascular Invasion and Herniation by HCC Quaglia et al 639

2 Table 1. Patient No. Age, y/sex Disease /M 62/F 47/M 58/F 42/M Patient Data and Results* ALD HBV HCV Cryptogenic HBV/HDV Size of Hepatocellular Carcinoma, mm * ALD indicates alcoholic liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; and HDV, hepatitis D virus. Main tumor mass recognizable macroscopically Size (or Size Range) of Intravascular Free-Floating Clusters 0.3 mm mm 0.3 mm 15 cells to 1 mm 3 cells to 2 mm Table 2. Details of Immunohistochemistry Methods* Reagent Source/Method Dilution Specificity Antifibrinogen Anti factor XIIIa Anti-CD34 Anti-SMA Dako BioGenex Dako Dako 1:2000 1:200 1:50 1:100 Fibrinogen, fibrinogen fragments D, E, X, Y Platelets Endothelium, hematopoietic progenitor cells Smooth muscle and myofibroblast * SMA indicates smooth muscle actin. Dako Corporation, Carpinteria, Calif; BioGenex, San Ramon, Calif. other markers, to demonstrate sinusoidal capillarization in nodular lesions in cirrhosis as part of our routine histologic assessment. 7 We have noted (unpublished observation) that the intravascular floating tumor clusters are covered by endothelium that stains for CD34. This observation has been noted in passing only rarely before The endothelial covering of intravascular HCC could act as a shield, protecting tumor clusters from activation of the coagulation cascade (which might in turn inhibit the metastatic process). Because the histologic identification of vascular invasion by HCC can be difficult, and because this feature can be an essential part of the diagnosis and prognostic evaluation of HCC, in this study we have explored ways of elucidating this matter with standard histochemical and immunohistochemical techniques. 11 We have investigated whether there is any evidence that vascular invasion (as defined at the histologic level by the presence of tumor clusters protruding or floating into vascular lumen) is the result of tumor destructive invasion; whether thrombus formation occurs or not; and whether the endothelial camouflage of tumor cells affects this process. The endothelial disguise could protect the HCC, and it hinders the detection of microvascular invasion by HCC, in the same fashion as a wolf in sheep s clothing. 12 MATERIALS AND METHODS We studied 5 cases of cirrhotic livers that contained a total number of 10 HCCs showing tumor clusters floating into the lumen of thin-walled vascular spaces in the vicinity of the main tumor. Information on patients, background liver, and tumors is given in Table 1. These cases were selected from our archive of livers removed at transplantation between 1997 and According to our records in the Department of Histopathology, during this period 81 transplants were carried out for cirrhosis. Hepatocellular carcinoma was present in 35 of these livers, 17 of which showed microscopic vascular invasion. In 7 of these cases, tumor clusters floating in the lumen of vessels were present. The 5 cases of this study were selected because they contained such tumor clusters floating into the lumen and because these clusters were present in consecutive sections to be used for special histochemical and immunohistochemical stains. Floating tumor clusters were defined as groups of tumor cells within a blood vessel, with at least three fourths of the circumference of the group detached from the vessel. Sections were used for staining with hematoxylin-eosin, and further consecutive sections were stained for fibrin (Martius scarlet blue [MSB], fibrinogen), platelets (factor XIIIa), smooth muscle actin, and endothelium (CD34), as described previously. 7,11 Details of the antibodies used and related methodology are given in Table 2. RESULTS These 5 cases were cirrhotic livers with a total number of 10 HCCs, with characteristics as shown in Table 1. All of the tumors were moderately differentiated HCCs. Definite intravascular HCC with associated thrombus was infrequent and was seen in only 1 HCC. Five of 10 HCCs that we studied showed the presence of tumor clusters floating in the lumen of thin-walled vascular structures (Table 1). In patient 1 the tumor was massive, composed of multiple nodules, and 1 intravascular floating tumor cluster was seen in a septum within the main tumor mass. In the other patients, these clusters were seen outside the main tumor mass, which was generally well demarcated by a rim of fibrous tissue, and we will refer to this rim as a fibrous capsule, despite the questionable applicability of this term in cirrhosis. 13 In all cases except 1, the tumor clusters were covered by endothelium, and no thrombus formation could be identified with MSB stain or by immunostaining for fibrinogen or factor XIII (Figure 1). In 1 case (patient 5), 2 tumor clusters showed an incomplete lining by endothelium, and on the surface of these clusters there was thrombus formation with fibrin and focal platelet aggregation (Figure 2). The presence of tumor clusters covered by endothelium without associated thrombus formation raises questions about the way tumor cells manage to float within vascular structures (without causing thrombosis). In 4 cases, a fibrous capsule (as described earlier) surrounded the main tumor mass. At low magnification, the capsule in many places was penetrated by tumor, which expanded laterally into the outer fibroconnective tissue giving a fungating appearance (Figure 3). A closer look at these areas of extratumoral expansion can show that the fronds of tumor outside the main mass and its fibrous capsule occupy the lumen of vascular channels and that intravascular free- 640 Arch Pathol Lab Med Vol 129, May 2005 Vascular Invasion and Herniation by HCC Quaglia et al

3 Figure 1. Patient 5. A free-floating tumor cluster shows no evidence of thrombus formation on hematoxylin-eosin stain (a, original magnification 100), with Martius scarlet blue staining (b, original magnification 100), or factor XIIIa immunostaining (c, original magnification 100). The cluster is completely covered by CD34-positive endothelium (d, original magnification 100). floating tumor clusters are present in the vicinity. The floating clusters usually appear to be detached from the fronds just described, but this could be due to cross-cutting of these fronds in the plane of section examined. In other words, a direct connection between these 2 structures cannot be excluded, because the apparently freefloating clusters may represent the tips of the neoplastic fronds of HCCs that have invaded the blood vessel wall to which the HCC is attached at another place. It was not possible to confirm this in all cases with serial sections because of the tortuous nature of the blood vessels involved and the indeterminate distance (into, or not represented in the tissue block) between the point of presumed entry of the HCC into the blood vessel and the plane of section studied. Alternatively, the free-floating HCC clusters could be tumor microemboli in transit or artifactual spillage of friable fragments from the main tumor mass. COMMENT Vascular invasion is generally considered to be a destructive process. Tumor cells infiltrate the stroma and vascular wall, disrupt the endothelium, and invade the vascular lumen. One would expect formation of thrombus at the site of vascular invasion as a result of the destruction of the vascular wall and endothelium. We have noted that HCC arising in a cirrhotic liver often shows tumor clusters occupying the lumen of vascular structures in the vicinity of HCC, but thrombus formation is rarely seen morphologically in association with these clusters, and moreover, these clusters are covered by endothelium. We have studied 10 HCCs in 5 cirrhotic livers, using histochemical and immunohistochemical staining to assess thrombus formation and its possible relationship with the endothelial covering of these tumor clusters. This observation has been noted in passing only rarely before in humans In a monkey model of HCC, Lindsay et al 14 noted that tumor spread in hepatic and pulmonary vessels was characterized by displacement of the vascular endothelial lining at the point of attachment of tumor to the vascular wall, with endothelium covering the luminal side of the tumor cluster. We found that the intravascular floating tumor clusters are covered by endothelium staining for CD34. Where this lining is intact, thrombus formation is not seen. In 1 case, Arch Pathol Lab Med Vol 129, May 2005 Vascular Invasion and Herniation by HCC Quaglia et al 641

4 Figure 2. Patient 5. One intravascular tumor cluster shows associated thrombus formation (arrows) on hematoxylin-eosin stain (a, original magnification 100), and with Martius scarlet blue staining (b, original magnification 200) and immunostaining for fibrinogen (c, original magnification 100). A discontinuous CD34-positive endothelial covering is present (d, original magnification 100, arrows). where the endothelial covering was partly disrupted, thrombus formation was evident. Therefore, in some instances, intravascular spread by HCC may not be the result of an invasive and destructive process, and we propose the following mechanism (Figure 4) as one of the possible ways by which HCC invades the vascular bed. Hepatocellular carcinoma is a richly vascular tumor with a complex, predominantly arterial supply and a complex network of capillarized sinusoids, 7,9,10,15 which is particularly evident in the HCCs with trabecular architecture. This complex sinusoidal network is in communication with vascular structures in the adjacent fibroconnective tissue. Eversion or herniation of tumor trabeculae through the peritumoral fibrous capsule may occur via these vascular channels forming everted (in a finger in glove fashion), frondlike protrusions that are covered by capillarized sinusoidal endothelium. These fronds of tumor protrude within the lumen of extratumoral vascular channels, and depending on the plane of section examined, they may appear as free-floating clusters. These clusters are still covered by endothelium, which acts as a shield, protecting tumor cells from the hematic environment and preventing thrombus formation. Rodent tail vein injection of single-cell carcinoma suspensions is associated with thrombus formation. 16 To expect thrombosis in the context of cirrhosis may seem paradoxical in view of the hypocoagulable state related to liver failure. However, portal vein thrombosis is frequently observed in cirrhotic livers, 17 and in our cases small fibrin thrombi could be detected in the background liver, and obliterated veins were seen in other parts of the specimens studied (not shown), indicating that functional microvascular coagulation was present in these cases. The presence of small thrombi in association with focal absence of endothelial covering suggests that some of these fronds of tumor are fragile and may detach from the intravascular neoplastic growth. This could be caused by blood flow related traumatic stress, mismatch between tumor growth and neoangiogenesis (tumor outgrowing the endothelium), tumor cells secreting substances that may digest the basal lamina and endothelium and trigger coagulation, or some combination of these causes. Adequate intravascular neoangiogenesis 14 and an effective anti- 642 Arch Pathol Lab Med Vol 129, May 2005 Vascular Invasion and Herniation by HCC Quaglia et al

5 Figure 3. Patient 3. Tumor eversion or herniation. The fungating appearance is caused by spread along a capsular vascular channel, which is still evident in one place (arrow). An apparently free-floating cluster (arrowhead) is seen in the vicinity, and the possibility that this cluster is connected to the area of herniation at a different level of the section cannot be excluded (a, Martius scarlet blue, original magnification 50; b, hematoxylineosin, original magnification 50). Figure 4. Proposed mechanism for vascular invasion and herniation. A, Hepatocellular carcinoma (HCC) surrounded by a fibrous capsule. Peritumoral vascular channels are in communication with the HCC capillarized sinusoidal network via intracapsular vascular spaces. B, Tumor herniates through these intracapsular vascular channels into peritumoral vascular spaces, expanding laterally. Increased intratumoral pressure could contribute to this process. C, These tumor fronds may be tortuous (*), and depending on the plane of section, they may appear as clusters of intravascular free-floating tumor (arrow). D, At some point, these fronds may start breaking up (because of shear stress, tumor outgrowth, etc), and thrombus formation begins to appear (yellow dots) where the endothelial lining is lost. Arteriovenous fistula may form at this stage. Arch Pathol Lab Med Vol 129, May 2005 Vascular Invasion and Herniation by HCC Quaglia et al 643

6 thrombotic endothelial coat would permit continued intravascular tumor growth and could be a prerequisite condition for tumors, such as HCC and renal carcinoma, that have a pronounced ability to grow along and survive within blood vessels, in continuity with the main tumor mass. The tumor will eventually fill the vascular lumen and further expand into the adjacent tissue, as demonstrated by Lindsay et al 14 in a monkey HCC model. These areas will then appear as encapsulated nodules of tumor, particularly if the vascular wall is not recognizable anymore. This could explain the presence of high-grade HCC deposits of relatively small size in cirrhotic livers 18 and also the fact that in our 5 cases, the largest tumors had a relatively small number of free-floating clusters and were characterized by multiple nodular deposits of tumor. These aspects need further investigation. A related matter concerns the nature of the HCC tumor capsule and the mechanism by which it is permeated. It may be that the herniation of HCC through the tumor capsule is a different process from capsular infiltration by tumor cells (although the distinction between these processes is difficult on morphologic grounds alone). The process could simply represent migration of tumor cells along lines of least mechanical resistance, like plants forcing their roots through soil, 6 and an intra/extratumoral pressure gradient may be at play. This hypothesis is consistent with the findings of Tanaka et al 19 in their study of tumor pressure in HCC. They found that HCC shows a positive tumor portal vein pressure gradient (mean pressure gradient 6 2H 2 O). These authors stress the role of the tumor portal vein pressure gradient and consider vascular invasion and generation of intrahepatic metastasis to be the result of pressure-driven dispersal of tumor cells into the portal vein. As Tanaka et al 19 point out, in more advanced HCC the tumor capsule tends to be destroyed by the infiltrative pattern of tumor cells. It is probably at this stage that destructive vascular invasion takes place, with stromal and vascular wall infiltration, disruption of the endothelium, and thrombus formation. 20,21 The angioarchitecture of HCC has been extensively investigated by Nakashima and Kojiro and their colleagues, 9,10,15 in their seminal work based on correlation of histology with postmortem arteriography and portography. In their detailed study of involvement of portal and hepatic veins by HCC, these authors describe fingerlike columns of tumor along portal veins, which may be classified into 4 types (ie, necrotic, proliferative, mixed, and organized). The proliferative type may be covered by endothelium, and shunt formation may occur between the arterial tumor circulation and the portal vein. Our findings are consistent with the original work of Nakashima and Kojiro and their colleagues. 9,10,15 We have observed communication between the intratumoral sinusoidal framework and the extratumoral vascular space (not shown). In the 5 cases we studied, we could not identify arterioles either within the free-floating intravascular clusters or at the point of herniation through the tumor capsule, but we cannot exclude the possibility that these protrusions may become arterialized. If this is the case, fragmentation of the intravascular fronds and rupture of arterioles could cause arteriovenous shunting, as described by Nakashima and Kojiro and their colleagues. 9,10,15 Vascular invasion is an important diagnostic and prognostic feature of HCC in cirrhosis. The lack of thrombosis associated with the unequivocal recognition of microvascular invasion by tumors generally may be explained by a process of endothelial sheltering, which may protect these HCC tumor projections from thrombus formation (like a wolf in sheep s clothing). The adoption by HCC of an endothelial coat could also act as a barrier to tumor extravasation, 22 causing a delay in seeding that might be exploited by intravascular drugs. 23 Further studies are needed to clarify whether different mechanisms of vascular spread may have different effects on survival and tumor recurrence, and whether this can be exploited from a therapeutic point of view. The authors wish to thank Mr Paul Bates and Miss Emma Bates for their kind assistance with the illustrations. References 1. Okuda K. Hepatocellular carcinoma. J Hepatol. 2000;32(suppl): Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular carcinoma: conclusions of the Barcelona-2000 EASL Conference. 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