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Dysplastic nodules and hepatocarcinogenesis
Clin Liver Dis 6 (2002) 497 – 512
Dysplastic nodules and hepatocarcinogenesis Neil D. Theise, MDa,*, Young Nyun Park, MDb, Masamichi Kojiro, MDc a
Department of Pathology, New York University Medical Center, Room 461, 560 First Avenue, New York, NY 10016, USA b Department of Pathology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea c Department of Pathology, Kurume University School of Medicine, Kurume, Japan
Recently, a growing literature has supported the concept that large nodules usually found in cirrhotic livers represent premalignant lesions in chronic liver disease. Using advanced imaging techniques in high-risk populations in Japan [1– 3], nodules suspicious for malignancy have often been identified and resected. Although some resected lesions were found to be small hepatocellular carcinomas (HCCs) [1,4,5], others were not. Some of these nonmalignant nodules were devoid of atypia; some had architectural or cytologic atypia insufficient for a diagnosis of HCC, although suggestive of a premalignant state; and others contained microscopic subnodules of HCC [1,4 – 7]. In follow-up studies from Japan, North America, Europe, and South Asia, including autopsy series [8– 10] and series of explants from liver transplant centers [11– 15], microscopic foci of HCC in the nodules were found and significant associations with HCC elsewhere in the same liver were established. These findings suggest that these nodular lesions are probably a frequent pathway of human hepatocarcinogenesis in a wide array of liver diseases and in diverse populations of patients. The emphasis in these early lesions has been somewhat different depending on the population of patients being studied. In Japan, where the bulk of available pathologic material comes from clinical biopsies and resections of lesions, more emphasis has been placed on higher-grade lesions and early HCCs, which are more likely to be detected radiologically and more closely associated with clinical progression, and therefore most important [14,16]. Outside of Japan, where large-scale screening of at-risk patients is not routine, most of the lesions have been identified and studied in the transplant setting, in whole cirrhotic livers. This source of pathologic material allows the physician to survey those lesions that
* Corresponding author. E-mail address: [email protected] (N.D. Theise). 1089-3261/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 3 2 6 1 ( 0 2 ) 0 0 0 0 6 - 5
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might not be detectable by conventional imaging but does not allow for follow-up data on the behavior of the lesions. Therefore, conclusions are based on associations, rather than observed progression.
Definitions and observations Dysplastic nodules consist of hepatocytes and almost always contain some intact portal tracts with a normal appearance. Because they are most often found in cirrhosis, they seem to correspond to what Edmondson called adenomatous hyperplasia (AH), although he viewed these lesions as having ‘‘limited growth potential’’ [17]. Other terms include macroregenerative nodule (MRN) [8,11, 12,15], dysplastic nodule [7], hepatocellular pseudotumor [13], and adenomatoid hyperplasia [14]. AH has been most widely used, though not exclusively, by researchers from Japan. MRN, named by Furuya et al [8] for the first Japanese autopsy study of the nodules, became the most widely accepted term in the earliest publications from North America and Europe. In 1997 a consensus document coauthored by investigators from Japan, North America, and Europe rejected all of these terms as being imprecise, if not actually misleading, and suggested the term dysplastic nodule, with further classification as low or high grade, to replace them [18]. Although researchers outside of Japan have generally accepted this new terminology, investigators in Japan have inconsistently adopted it, usually continuing to use the term AH. This review uses dysplastic nodule (DN) as the preferred term, recognizing that it, too, is problematic. DNs are defined grossly as large hepatic nodules that are distinct from the surrounding liver parenchyma in terms of size, color, texture, or the degree to
Fig. 1. Low-grade dysplastic nodule (DN). In this resection from a patient with primary biliary cirrhosis, a nodule distinct from surrounding nodules in size is easily seen; however, confirmation requires histologic examination.
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which they bulge from the cut surface of the liver (Fig. 1). A nodule is confirmed as a DN with histologic examination and the identification of intact portal structures distributed through the lesion. The number of these portal structures may be mildly or greatly reduced compared with a similar area of nondiseased hepatic parenchyma [19]. DNs have been found in a wide variety of chronic liver diseases [11,12,15], including processes that are hepatitic (hepatitis B, C, and autoimmune hepatitis), cholangitic (primary biliary cirrhosis, primary sclerosing cholangitis), metabolic (a-1-antitrypsin deficiency, primary hemochromatosis), and toxic (alcoholic liver injury). Typically, livers with DNs contain a small number of these nodules, rarely more than 10, although there are exceptions that are discussed later [8– 12,15]. DNs may be subclassified as low or high grade according to features listed in Table 1. Low-grade lesions may be devoid of atypia or display features of largecell change. They may be iron or copper retentive or they may be diffusely steatotic. They should not contain nodule-in-nodule – type lesions or small-cell change, or features closely associated with HCC; these changes would indicate a high-grade lesion. It is usually not possible to radiographically distinguish small HCCs from DNs or high-grade DNs from low-grade DNs with complete confidence [20,21], nor is it usually possible to reliably make such distinctions on the basis of gross morphology. Histologic examination, either by biopsy or examination of a resected specimen, is required for accurate classification.
Histologic features of low-grade DNs Low-grade DNs are well-defined nodules surrounded by a condensed rim of fibrous tissue similar to that surrounding cirrhotic nodules (Fig. 2). The nodules are therefore not truly encapsulated. Portal tracts, present in virtually all DNs, are most often uniformly distributed in low-grade DNs and may even be distributed in a virtually normal fashion with regularly intervening terminal hepatic venules. In
Table 1 Features that may be found in low-grade (ordinary) and high-grade (atypical) dysplastic nodules Low-grade DNs
High-grade DNs
Normal architecture Normal cytology or large-cell change Iron accumulation in otherwise nonsiderotic liver Iron resistance in otherwise siderotic liver Diffuse fatty change Copper accummulation Schirrous growth in the absence of other atypia
Focal pseudogland formation Diffuse small-cell change Nodule-in-nodule lesions with small-cell change Iron accumulation Iron resistance in siderotic nodule Fatty change Clear cell change Mallory body clustering Decreased reticulin fibers Features diagnostic of hepatocellular carcinoma
Abbreviation: DN, dysplastic nodule.
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Fig. 2. Low-grade dysplastic nodule. Microscopic examination of a distinctly large nodule (left of f ibrous septum) in hepatitis C cirrhosis confirms the absence of cytologic or architectural atypia (hematoxylin-eosin, original magnification 20).
some nodules, portal structures may be caught up in fibrous septa, which partially subdivide the nodule. The hepatocytes of low-grade DNs tend to be comparable in size to hepatocytes outside the lesion. The hepatocytes may display changes characteristic of the underlying liver disease affecting the surrounding liver, such as fatty change, Mallory bodies, or increased iron or copper deposition. These changes will be distributed in the DN as they are in surrounding cirrhotic nodules. Occasionally, a DN in a nonsiderotic liver may contain increased iron or a DN in an otherwise siderotic liver will be iron free; however, it would still be classified as low grade. Rarely, a similar increase in copper, in an otherwise copper-free liver, is also seen. These changes in DNs, when not confined to a subnodule within the lesion (ie, copper or iron retention and fat accumulation), may represent a marker of the clonality in the DN hepatocytes. Studies of large-cell change in cirrhosis and statistical evaluation of the association of this feature in DNs and HCCs [12,15] indicate the likelihood that large-cell change is usually a reactive, not a premalignant, change (although it also may serve as a marker for livers at increased risk for HCC). Therefore, it has been recommended that DNs containing large-cell change without other atypical features be classified as low-grade lesions [12]. Scirrhous changes, stellate fibrosis of DN portal tracts, or diffuse pericellular fibrosis in regions of DN parenchyma have previously been thought to be features of high-grade DNs; however, careful analysis of scirrhous change shows that it does not segregate with other features of high-grade lesions and appears in otherwise low- or high-grade lesions equally (Author’s unpublished data, N.D.T. 1999). Therefore, we would recommend that scirrhous change be excluded from the diagnostic criteria for high-grade DNs.
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Histologic features of high-grade DNs High-grade DNs are defined by the presence of small-cell change and/or architectural atypia. They are usually well circumscribed and surrounded by a condensed rim of fibrous tissue, as in low-grade lesions, although some may merge focally with adjacent liver parenchyma [22]. The atypical features in high-grade DNs may take a variety of forms and may be diffuse throughout the nodule or focal. Diffuse changes most often fall into the category of cellular atypia. The definition of cellular atypia in this setting should be limited to small-cell change: small, crowded hepatocytes with basophilic cytoplasm and an increased nuclear to cytoplasmic ratio [23]. Small-cell change appears to be consistently related to the development of HCC in a variety of studies and should remain a criterion for an atypical designation. This cellular feature is reported more frequently in studies from Japan than those from other countries [8– 12,15]; this discrepancy remains unexplained. Pseudoacinar structures resembling those seen in well-differentiated HCCs are a form of architectural atypia in high-grade DNs and may be either focal or diffuse. Focal atypia may merge with the surrounding DN parenchyma, but it more often occurs as a nodule-in-nodule lesion (Fig. 3) [12]. Such subnodules often appear to compress the adjacent DN parenchyma, and studies of the proliferation rates of the cells making up these lesions indicate that they are proliferating more rapidly than the surrounding tissue [24]. These subnodules may display small cell change (SCC) [15,23] but may also show changes that are not classically atypical, including fatty change [25], clear cell change [15,20], clusters of hepatocytes with Mallory’s hyaline [26], increased iron uptake within the DN [27], iron resistance in an otherwise siderotic nodule [27,28], and accumulation of copper-
Fig. 3. Small-cell change in a high-grade dysplastic nodule. Hepatocytes demonstrating small-cell change have hyperchromatic nuclei, nearly normal in size, with reduced, often basophilic, cytoplasm, thus an increased cytoplasmic ratio. Hepatocyte plates in small-cell change are often thickened or have a cobblestone appearance (upper right; hematoxylin-eosin, original magnification 40).
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Fig. 4. High-grade dysplastic nodule (DN) and hepatocellular carcinoma (HCC). DNs (open arrow) are often found in association with HCC (solid arrow) elsewhere in the liver. This patient with hepatitis B cirrhosis was found to have two nodules on imaging and underwent resection.
binding protein [29]. Some expansile subnodules do not display any distinctive cytologic features, although, architecturally, they may display a pseudoacinar growth pattern. Therefore, all subnodules, with or without distinctive cellular changes, are appropriately defined as architectural atypia on the basis of the expansile growth and should warrant classification of the entire DN as a highgrade lesion [12]. HCC may be identified in high-grade DNs (Fig. 4) [1,5,7 – 13,15,29 – 32]. These HCC microfoci may display any of the features seen in larger HCCs, although they are usually well differentiated. Typical growth patterns include pseudoacinus formation, thickened trabeculae, or a scirrhous growth. Common cytologic features include intracytoplasmic Mallory’s hyaline, fatty change, clear cell change, iron resistance, and multinucleation. Multiple foci of HCC may also be found in a single DN and the histologic features of these foci are often different from each other [30,32]. The DN parenchyma surrounding a microfocus of HCC will usually contain portal tracts and may consist of seemingly normal hepatocytes, suggesting a background of a low-grade MRN, or it may show atypia, indicating a high-grade background. Either way, by convention, DNs containing foci of HCC are classified as high grade.
The premalignant nature of DNs The association of DNs with HCC is demonstrated in two ways. First, as mentioned previously, DNs sometimes contain one or multiple microscopic foci of HCC. Second, DNs are sometimes found in livers that also contain grossly apparent HCC elsewhere (see Fig. 1). These relationships have been found to
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achieve statistical significance [11,12,15]. In addition to this statistical correlation, the atypical features often found in DNs include many that are thought to be premalignant. The clustering of hepatocytes containing Mallory’s hyaline [33,34] and foci of iron resistance in siderotic nodules [28,35] have been independently described as premalignant changes. Small cell change, considered premalignant on the basis of morphometric analysis, also has been seen and, in some series, is very common [5,10 –13,15,18]. Other features that are commonly identified in mature HCC are found in highgrade DNs. For example, immunohistochemical studies of DN sinusoids reveal increasing degrees of capillarization (ie, loss of endothelial fenestration, deposition of basement membrane, and expression of antigens such as factor VIII and CD34) with the development of atypia and HCC [36,37]. Studies of ploidy indicate an increased frequency of aneuploidy in high-grade DNs [38,39]. Immunohistochemical staining for a-fetoprotein [13,40,41] and p-Ras [13] demonstrates expression of these proteins in atypical foci and microfoci of HCC. Clonal stepwise progression has also been demonstrated in a subnodule of HCC arising in a DN by Tsuda et al [42]. Their study exploited integration of the hepatitis B surface antigen gene into host hepatocytes using restriction length polymorphism analysis, demonstrating that a microfocus of HCC consisted of cells derived from the same clone as the surrounding DN parenchyma. These findings represent static samplings of the process of malignant transformation. The association of DNs with HCC elsewhere in the same liver shows that DNs are at least a marker of a liver with a tendency to generate malignancies. Several studies, however, now demonstrate that, when followed over time by serial biopies or biopsy followed by radiographic changes indicative of expansile growth, there is progression from at least high-grade DN to HCC [4,5,43 –45]. In the previously described longitudinal studies, low-grade DNs are not as obviously premalignant or as indicative of neighboring malignant transformation as are high-grade DNs. The data addressing this difference appear contradictory. Studies of the various aspects of DNs mentioned—proliferation [13,40,46 – 48], p-Ras expression [13], ploidy [38,39], and endothelial and extracellular matrix alterations [36,37,49]—found similarities between high-grade DNs and HCC. In the absence of these similarities between low-grade DNs and HCC, most of these researchers concluded that high-grade lesions are related to HCC, whereas lowgrade lesions are related to regenerative nodules and are less clearly implicated in hepatocarcinogenesis. Data from the largest series of liver explants support and contradict this view of low-grade lesions [12]. A subset of livers was identified (13 of 155) that contained so many DNs as to be virtually uncountable. When large LCD was excluded as a criterion for atypia, the sampled DNs were low grade in all but one of these livers. None of these livers contained HCC. All such livers were in patients with chronic hepatitis (hepatitis B, hepatitis C, or autoimmune hepatitis), and the mean age of these patients was significantly lower than the other patients with few or no DNs. These findings suggest that these DNs are actually large regenerative nodules that have not yet scarred down into smaller nodules, and therefore examples of AH
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rather than neoplastic lesions. These DNs might also correlate with large cirrhotic nodules that became undetectable on long-term ultrasound surveillance in a study by Kondo et al [50]. However, statistical associations of low-grade lesions with coexistent HCC in this, as in earlier, series are strong, even when high-grade lesions are excluded from analysis [12,15]. Thus, the neoplastic, premalignant nature of low-grade lesions seems more open to question than it does for high-grade lesions, although perhaps most researchers (this study’s authors included) favor a distinction between these lesions. An explanation for the discrepancies, on the basis of hepatic stem cells, possibly resolves the apparent contradictions [51].
Speculations on the early stages of human hepatocarcinogenesis How DNs actually form has not yet been firmly established. One early view suggested that an ordinary regenerative nodule in cirrhosis becomes more rapidly proliferative, therefore larger. In turn, with the increased proliferation, it also becomes at greater risk for the carcinogenic ‘‘hits,’’ thereby giving rise to atypia and carcinoma [6]. Although simple, this hypothesis does not take into consideration three known facts about DNs. First, they can be found in livers in advance of cirrhosis and therefore do not always arise from a preexistent regenerative nodule [8,32,52]. Second, the presence of many intact portal tracts in most DNs, which have not yet been demonstrated to fully reconstitute after scarring and injury, suggests that they must preexist the formation of the DN, making it unlikely that a small cirrhotic nodule with few if any portal tracts could enlarge to a nodule with many portal tracts. Third, some DNs are clonal lesions, not hyperplastic phenomena [42,53 – 55]. To account for these features, studies have suggested an alternative process of DN development [32,51,56]. This alternate hypothesis has been able to predict some previously unexplored features of DNs and, perhaps, can be broadened to explain other types of borderline or early carcinomatous features. The steps of this hypothesis are as follows: 1.
2. 3.
4.
A clonal expansion of hepatocytes follows on the earliest carcinogenic events in response to any diffuse injury of the, liver which leads to increased hepatocyte turn over. These early hits lead to a clonal expansion of hepatocytes, which spread around adjacent portal structures rather than displacing them. As the rest of the liver becomes scarred, progressing to later stages of disease and eventually cirrhosis, the island of clonal hepatocytes, if resistent to the scarring affecting the rest of the liver, would remain intact— an island of relatively preserved hepatic parenchyma made up of neoplastic, clonal hepatocytes. With establishment of cirrhosis in the adjacent liver, the clonal expansion takes on the appearance of a large cirrhotic nodule.
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Having already undergone the earliest transforming events of hepatocarcinogenesis, the clonal, hepatocyte expansion remains at increased risk for later developments and thus the lesion becomes the likeliest site of full malignant transformation.
This hypothesis suggested a number of testable predictions that have resulted in supportive studies. First, expansion of a clonal population as described would not necessarily depend on rapid proliferation but might arise from a relatively low proliferation rate balanced by inhibition of apoptosis. In fact, many studies of proliferation have confirmed that the low-grade DNs and the background parenchyma of nodule-in-nodule – type high-grade DNs are no more proliferative, and are often less proliferative, than surrounding cirrhotic nodules [13,24,46 –48]. Of course, if these hepatocytes had a survival advantage compared with surrounding nonneoplastic hepatocytes, they would expand in a clustered fashion. One possibility is that they might be resistant to the disease damaging the surrounding parenchyma (eg, inability to be virally infected, absence of antigen presentation once infected, etc.) or they might have impaired mechanisms of apoptosis. We tested this latter possibility and found that apoptosis did appear diminished in low-grade DNs, only increasing with the emergence of atypia [57]. The rates of apoptosis in low-grade DNs were not significantly lower than those of surrounding regenerative nodules; however, the ratio of apoptosis to proliferation was increased in regenerative nodules, indicating that the hepatocyte populations, as expected, had a survival advantage when compared with non-DN hepatocytes. A key feature of our hypothesis of DN development is that the nodular appearance arises not from expansile growth, but because the clonal hepatocytes expand with reduced or absent scarring, while the surrounding, nonneoplastic liver becomes cirrhotic. Thus, the authors made a second testable prediction, which was confirmed: that there would be diminished stellate cell activation in DNs [58]. Activated stellate cells, shown by immunohistochemical staining for smooth muscle actin, were significantly fewer within DNs than in the surrounding cirrhotic nodules. To the authors’ knowledge, no other studies of this aspect of DN pathophysiology have been reported. Finally, clonality of many, if not all, DNs has been demonstrated in many studies [42,53 –55,59,60]. Many nodules that appear to regenerate have also been identified as clonal expansions, and neighboring, clustered, smaller cirrhotic nodules are often of the same clone [61]. This suggests, again, that the clonal expansion preceded the development of cirrhosis and that scarring subsequently subdivided the clonal expansion into separate nodules. Moreover, clonality of fields of hepatocytes has been identified in advance of cirrhosis, even in normal liver tissue [54]. Studies on the loss of heterozygosity have shown similar results [59].This suggests that some clonality is not related to neoplastic transformation but is a product of liver tissue formation in embryogenesis. However, these data confirm that clonal expansions can occur without displacing original portal structures, as predicted in the previously described model of DN development. How this precedes architecturally remains unclear. But, in animal models of
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Fig. 5. Hepatocellular carcinoma (HCC) in a dysplastic nodule (DN). (A) A nodule-in-nodule growth pattern (arrowhead) in a DN (hematoxylin-eosin, original magnification 10). (B) View of a region in Fig. 5A shows a moderately differentiated HCC in a background of otherwise unremarkable hepatocytes (hematoxylin-eosin, original magnification 40). (See also Color Plate 12.)
hepatocyte transplantation in which a small population of transplanted cells completely repopulates a genetically damaged liver, such as in the tyrosinemia mice of Markus Grompe et al [62], complete repopulation around native structures has been demonstrated.
Fig. 6. Multiple, small hepatocellular carcinomas (HCCs) in adjacent cirrhotic nodules in a patient with chronic hepatitis C; many of these nodules contain de novo HCC. The great variability of histologies in the HCCs strongly suggests that at least some of them are independent primaries, rather than invasion or metastasis into adjacent nodules by a single malignant clone. No HCC was identified in any other region of this explanted liver (Trichrome, original magnification 2). (See also Color Plate 13.)
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Toward a general concept of hepatocarcinogenesis Thorough, routine examination of cirrhotic liver explants inevitably will yield some early HCCs that do not appear to arise within a demonstrable DN. These are relatively infrequent and fall into two basic categories. The first is an HCC arising in a cirrhotic nodule that is not distinctive in any way, not differing from its neighbors in color, texture, bulging, or size (ie, what might be called a ‘‘de novo’’ HCC). This is easy to contain within our concept of DN, simply showing up our inability to distinguish neoplastic nodules unless they supply us with a clear morphologic marker of their distinctiveness. Such small cirrhotic nodules with HCCs might simply be small, low-grade DNs, without the size or accumulation of pigment to be noticable. The second category of small HCCs found incidentally in explants is rarer still. This category consists of multiple, very small HCCs, often with distinct morphologies and patterns of growth suggesting that they are independent lesions, arising in a discrete cluster of neighboring, small, cirrhotic nodules in a liver otherwise devoid of HCC (Fig. 5). Although no background DN is identified in these lesions, they likely represent the same process of HCC development from a prior clonal expansion as postulated for DN-related carcinogenesis. The clustering of so many nodules in a discrete area suggests that there is something distinctive about those clustered nodules. The comparison of this type of lesion to the clustered cirrhotic nodules of identical clones lends support to the idea that the clonality is the important feature, the appearance as a DN is merely an artifact of suppression of hepatic stellate cell (HSC) activation. Clonal expansions in which HSC activation occurs at normal levels in response to injury produce such clonal clusters and, just like DNs, these can give rise to HCC. We may therefore suggest a broader conception of hepatocarcinogenesis as portrayed in Fig. 7.
Fig. 7. Pathways of hepatocarcinogenesis. HSC = hepatic stellate cell; DN = dysplastic nodule; HCC = hepatocellular carcinoma.
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Areas of diagnostic uncertainty The generalized concept of early hepatocarcinogenesis can help to reconcile lesions, most often described by Japanese investigators, that do not match the classic descriptions of DNs. Lesions described as ‘‘early hepatocellular carcinoma’’ [63 –66], which often have indistinct borders or are identified in the absence of fully established cirrhosis, may simply reflect differing stages of scarring within the neoplastic expansions as opposed to the surrounding liver. The indistinct nature of the lesions suggests that they are a different neoplastic pathway; however, they may arise from a similar process (see Fig. 5). More difficult is the decision of where to draw the diagnostic line between dysplasia and carcinoma. An informal comparison of diagnoses for a range of hepatocellular neoplastic lesions among Japanese and non-Japanese pathologists (International Consensus Panel for Hepatocellular Carcinoma, Kurume, Japan, 2002, M.K., unpublished data) shows a lack of consistency within and between those two groups when it comes to deciding ‘‘cancer’’ versus ‘‘not yet cancer.’’ The type of lesion most difficult to diagnose is shown in Fig. 6. However, there is consensus among the reviewing pathologists on the more established carcinomas and low-grade lesions. Not surprisingly, then, the ‘‘borderline’’ category is the current area of controversy and leads to the greatest confusion when trying to compare studies from different investigators. Additional uncertainty in nodule characterization lies in the redundancy of so many adaptive or pathologic changes in the liver, with each of many morphologic changes arising from different causes. For example, fatty change in DNs or early HCC may arise from clonal changes in lipid metabolism or sensitivity to alcohol toxicity, or to changes in the ratio of arterial to portal blood flow, as the latter is lost during the neoplastic progression [64]. Mallory bodies may also represent a clonal marker of neoplastic alterations in gene expression, but could arise from chronic cholestasis in a nodule with incomplete biliary drainage, or an altered response to alcohol or lipid metabolism. Hepatocytes within portal tract or septal stroma may represent invasion [65 – 67], entrapment of nonneoplastic hepatocytes by scarring, ‘‘regression of cirrhosis’’ as described by Wanless et al [68], or hepatocyte regeneration from stem cells [69]. Thus, use of any of these histologic features for diagnostic purposes may be tremendously imprecise; each may indicate different things in different settings, such as evidence of clonality and neoplasia or evidence of simple regeneration.
Summary In the last decade, careful examination of explanted cirrhotic livers in liver transplant centers around the world has confirmed the findings of the earlier Japanese investigators: DNs (by this or any other name) represent hepatic, premalignant lesions in chronic liver disease. Careful examination of their gross and microscopic morphologies has led to the hypothesis of precirrhotic, spreading
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clonal expansions that are resistent to scarring, and that result in neoplastic islands of hepatic parenchyma. The resultant distinctive nodules, often marked by features suggestive of their clonality (such as increased pigment), are at increased risk for subsequent carcinomatous events, thereby giving rise to HCC. Specialized molecular and immunohistochemical studies confirm many aspects of this hypothesis. In suggesting that some aspects of DN pathophysiology are not integral to the carcinogenetic pathway (ie, inhibition of HSC inactivation), this hypothesis serves a broader purpose, explaining the various settings in which early HCCs are found in cirrhotic explants and in wedge resections of radiographically defined lesions. Discrepancies between Japanese and non-Japanese investigations regarding dysplasia and early HCCs reflect not different biologic pathways but differences in detection, interpretation, and application of nomenclature. These differences may fade away as more international collaborative work brings investigators of diverse nationalities into regular contact, supporting movement toward a commonly acceptable nomenclature and set of diagnostic criteria. Ultimately, an understanding of the pathophysiology of these lesions, through more detailed molecular and physiologic studies, should lead to more efficient and available early detection, and perhaps chemoprevention approaches to hepatic malignancy.
References [1] Arakawa M, Kage M, Sugihara S, Nakashima T, Suenaga M, Okuda K. Emergence of malignant lesions within an adenomatous hyperplastic nodule in a cirrhotic liver: observation in five cases. Gastroenterology 1986;91:198 – 208. [2] Okuda K. Early recognition of hepatocellular carcinoma. Hepatology 1986;9:751 – 5. [3] Shinagawa T, Ohto M, Kimura K, Tsunetomi S, Morita M, Saisho H, et al. Diagnosis and clinical features of small hepatocellular carcinoma with emphasis on the utility of real-time ultrasonography. A study in 51 patients. Gastroenterology 1984;86:495 – 502. [4] Kaji K, Terada T, Nakanuma Y. Frequent occurrence of hepatocellular carcinoma in cirrhotic livers after surgical resection of atypical adenomatous hyperplasia (borderline hepatocellular lesion): a follow-up study. Am J Gastroenterol 1994;89:903 – 8. [5] Takayama T, Makuuchi M, Hirohashi S, Sakamoto M, Okazaki N, Takayasu K, et al. Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 1990;336: 1150 – 3. [6] Sakamoto M, Hirohashi S, Shimosato Y. Early stages of multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol 1991;22:172 – 8. [7] Wada K, Kondo Y, Kondo F. Large regenerative nodules and dysplastic nodules in cirrhotic livers: a histopathologic study. Hepatology 1988;8:1684 – 8. [8] Furuya K, Nakamura M, Yamamoto Y, et al. Macroregenerative nodule of the liver. A clinicopathological study of 345 autopsy cases of chronic liver disease. Cancer 1988;61:99 – 105. [9] Terada T, Terasaki S, Nakanuma Y. A clinicopathologic study of adenomatous hyperplasia of the liver in 209 consecutive cirrhotic livers examined by autopsy. Cancer 1993;72:1551 – 6. [10] Vaiphei K, Ghosh UK, Vasishta RK. Incidence of adenomatous hyperplasia in postmortem cirrhotic livers and study of cellular proliferative indices by light microscopy. Indian J Gastroenterol 1999;18:7 – 10. [11] Ferrell L, Wright T, Lake J, et al. Incidence and diagnostic features of macroregenerative nodules vs small hepatocellular carcinoma in cirrhotic livers. Hepatology 1992;16:1372 – 81.
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[12] Hytiroglou P, Theise ND, Schwartz M, Mor E, Miller C, Thung SN. Macroregenerative nodules in a series of adult cirrhotic liver explants: issues of classification and nomenclature. Hepatology 1995;21:703 – 8. [13] Le Bail B, Belleannee G, Bernard P-H, Saric J, Balabaud C, Bioulac-Sage P. Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 lesions in the cirrhotic explants of 10 adult French patients. Hum Pathol 1995;26:897 – 906. [14] Mion F, Grozel L, Boillot O, Paliard P, Berger F. Adult cirrhotic liver explants: precancerous lesions and undetected small hepatocellular carcinomas. Gastroenterology 1996;111:1587 – 92. [15] Theise ND, Schwartz M, Miller C, Thung SN. Macroregenerative nodules and hepatocellular carcinoma in forty-four sequential adult liver explants with cirrhosis. Hepatology 1992;16: 949 – 55. [16] Nakashima O, Sugihara S, Kage M, Kojiro M. Pathomorphologic characteristics of small hepatocellular carcinoma: a special reference to small hepatocellular carcinoma with indistinct margins. Hepatology 1995;22:101 – 5. [17] Craig JR, Peters RL, Edmondson HA. Tumours of the liver and intrahepatic bile ducts. Washington, DC: AFIP; 1989. [18] Wanless I, Callea R, Craig JR, et al. Terminology of nodular lesions of the liver: recommendations of the World Congress of Gastroenterology Working Group. Hepatology 1995;22: 983 – 93. [19] Nakashima Y, Nakashima O, Hsia CC, Kojiro M, Tabor E. Vascularization of small hepatocellular carcinomas: correlation with differentiation. Liver 1999;19:12 – 8. [20] Krinsky G, Theise ND, Rofsky N, Mizrachi H, Tepperman LW, Weinreb JC. Dysplastic nodules: arterial phase enhancement at CT and MR imaging with whole explant pathologic correlation—a case report. Radiology 1998;209:461 – 4. [21] Krinsky G, Lee VS, Theise N. Focal lesions in the cirrhotic liver—high resolution ex-vivo MR imaging with pathologic correlation. J Comput Assist Tomogr 2000;24:189 – 96. [22] Nakanuma Y, Terada T, Terasaki S, Ueda K, Nonomura A, Kawahara E, et al. ‘‘Atypical adenomatous hyperplasia’’ in liver cirrhosis: low grade hepatocellular carcinoma or borderline lesion? Histopathology 1990;17:27 – 36. [23] Watanabe S, Okita K, Harada T, Kodama T, Numa Y, Takemoto T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;55:2197 – 205. [24] Theise ND, Marcelin K, Goldfischer M, Hytiroglou P, Ferrell L, Thung SN. Low proliferative activity in macroregenerative nodules: evidence for an alternate hypothesis concerning human hepatocarcinogenesis. Liver 1996;16:134 – 9. [25] Terada T, Nakanuma Y, Hoso M, Saito K, Sasaki M, Nonomura A. Fatty macroregenerative nodule in non-steatotic liver cirrhosis: a morphologic study. Virchows Arch A Pathol Anat Histopathol 1989;415:131 – 6. [26] Terada T, Hoso M, Nakanuma Y. Mallory body clustering in adenomatous hyperplasia in human cirrhotic livers: report of four cases. Hum Pathol 1989;20:886 – 90. [27] Terada T, Nakanuma Y. Survey of iron-accumulative macroregenerative nodules in cirrhotic livers. Hepatology 1989;5:851 – 4. [28] Terada T, Nakanuma Y. Iron-negative foci in siderotic macroregenerative nodules in human cirrhotic livers: a marker of incipient pre-neoplastic or neoplastic lesions. Arch Pathol Lab Med 1989;113:916 – 20. [29] Nakanuma Y, Terada T, Ueda K, Terasaki S, Nonomura A, Matsui O. Adenomatous hyperplasia of the liver as a precancerous lesion. Liver 1993;13:1 – 9. [30] Ohno Y, Shiga J, Machinami R. A histopathological analysis of five cases of adenomatous hyperplasia containing minute hepatocellular carcinoma. Acta Pathol Jpn 1990;40:267 – 78. [31] Sakurai M, Wakasa K, Monden M, Yamada T, Kuroda C, Marukawa T, et al. Hepatocellular carcinoma in adenomatous hyperplasia of the liver. Cancer Chemother Pharmacol 1989; 23(Suppl):S110 – 3. [32] Theise ND, Lapook JD, Thung SN. A macroregenerative nodule containing multiple foci of hepatocellular carcinoma in a non-cirrhotic liver. Hepatology 1993;17:993 – 6.
N.D. Theise et al. / Clin Liver Dis 6 (2002) 497–512
511
[33] Nakanuma Y, Ohta G. Small hepatocellular carcinoma containing many Mallory bodies. Liver 1984;4:128 – 33. [34] Nakanuma Y, Ohta G. Is Mallory body formation a preneoplastic change? A study of 181 cases of liver bearing hepatocellular carcinoma and 82 cases of cirrhosis. Cancer 1984;55:2400 – 4. [35] Deugnier Y, Charalambous P, le Quilleuc D, Turlin B, Searle J, Brissot P, et al. Preneoplastic significance of hepatic iron-free foci in genetic hemochromatosis: a study of 185 patients. Hepatology 1993;18:1363 – 9. [36] Dhillon A, Colombari R, Savage K, Scheuer PJ. An immunohistochemical study of the blood vessels within primary hepatocellular tumors. Liver 1992;12:311 – 8. [37] Terada T, Nakanuma Y. Expression of ABH blood group antigens, receptors of Ulex europaeus agglutinin I, and factor VIII – related antigen on sinusoidal endothelial cells in adenomatous hyperplasia in human cirrhotic livers. Hum Pathol 1991;22:486 – 93. [38] Hoso M, Nakanuma Y. Cytophotometric DNA analysis of adenomatous hyperplasia in cirrhotic livers. Virchows Arch A 1991;418:401 – 4. [39] Orsatti G, Theise ND, Thung SN, Paronetto F. DNA image cytometric anlaysis of macroregenerative nodules (adenomatous hyperplasia) of the liver: evidence in support of their preneoplastic nature. Hepatology 1993;17:621 – 7. [40] Terasaki S, Terada T, Nakanuma Y, Nonomura A, Unoura M, Kobayashi K. Argyrophilic nucleolar organizer regions and alpha-fetoprotein in adenomatous hyperplasia in human cirrhotic livers. Am J Clin Pathol 1991;95:850 – 7. [41] Theise N, Fiel I, Hytiroglou P, Ferrell L, Schwartz M, Miller C, et al. Macroregenerative nodules in cirrhosis are not associated with elevated serum or stainable tissue alpha-fetoprotein. Liver 1994;15:30 – 4. [42] Tsuda H, Hirohashi S, Shimamoto Y, Terada M, Masegawa H. Clonal origin of atypical adenomatous hyperplasia and clonal identity with hepatocellular carcinoma. Gastroenterology 1998;95:1664 – 6. [43] Lencioni R, Caramella D, Bartolozzi C, Di Coscio G. Long-term follow-up study of adenomatous hyperplasia in liver cirrhosis. Ital J Gastroenterol 1994;26:163 – 8. [44] Sakamoto M, Hirohashi S. Natural history and prognosis of adenomatous hyperplasia and early hepatocellular carcinoma: multi-institutional analysis of 53 nodules followed up for more than 6 months and 141 patients with single early hepatocellular carcinoma treated by surgical resection or percutaneous ethanol injection. Jpn J Clin Oncol 1998;28:604 – 8. [45] Seki S, Sakaguchi H, Kitada T, Tamori A, Takeda T, Kawada N, et al. Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study. Clin Cancer Res 2000;6:3469 – 73. [46] Grigioni W, D’Errico A, Bacci F, Gaudio M, Mazziotti A, Gozzetti G, et al. Primary liver neoplasms: evaluation of proliferative index using MoAb Ki67. J Pathol 1989;158:23 – 9. [47] Terada T, Nakanuma Y. Cell proliferative activity of adenomatous hyperplasia in the liver and small hepatocellular carcinoma. An immunohistochemical study demonstrating proliferating cell nuclear antigen. Cancer 1992;70:591 – 8. [48] Tiniakos DG, Brunt EM. Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study. Liver 1999;19:58 – 68. [49] Park YN, Yang CP, Fernandez GJ, Cubukcu O, Thung SN, Theise ND. Neoangiogenesis and sinusoidal ‘‘capillarization’’ in dysplastic nodules of the liver. Am J Surg Pathol 1998;22:656 – 62. [50] Kondo F, Ebara M, Sugiura N, Wada K, Kita K, Hirooka N, et al. Histological features and clinical course of large regenerative nodules: evaluation of their precancerous potentiality. Hepatology 1990;12:592 – 8. [51] Theise N, Hytiroglou P, Thung S. Macroregenerative nodules and hepatocarcino-genesis. Il Friuli Medico Alpe Adria J Med 1994;49:637 – 50. [52] Matsuno Y, Hirohashi S, Furuya S, Sakamoto M, Mukai K, Shimosato Y. Heterogeneity of proliferative activity in nodule-in-nodule lesions of small hepatocellular carcinoma. Jpn J Cancer Res 1990;81:1137 – 40. [53] Aihara T, Noguchi S, Sasaki Y, Nakano H, Monden M, Imaoka S. Clonal analysis of precancerous lesion of hepatocellular carcinoma. Gastroenterology 1996;111:455 – 61.
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N.D. Theise et al. / Clin Liver Dis 6 (2002) 497–512
[54] Ochiai T, Urata Y, Yamano T, Yamagishi H, Ashihara T. Clonal expansion in evolution of chronic hepatitis to hepatocellular carcinoma as seen at an X-chromosome locus. Hepatology 2000;31:615 – 21. [55] Paradis V, Laurendeau I, Vidaud M, Bedossa P. Clonal analysis of macronodules in cirrhosis. Hepatology 1998;28:953 – 8. [56] Theise ND. Macroregenerative (dysplastic) nodules and hepatocarcinogenesis: theoretical and clinical considerations. Semin Liver Dis 1995;15:360 – 71. [57] Park YN, Chae KJ, Kim YB, Park C, Theise ND. Increased activity of apoptosis and proliferation in human hepatocarcinogenesis. Cancer 2001;92:2733 – 8. [58] Park YN, Yang CP, Cubukcu O, Thung SN, Theise ND. Hepatic stellate cell activation in dysplastic nodules: evidence for an alternate hypothesis concerning human hepatocarcinogenesis. Liver 1997;17:271 – 4. [59] Maggioni M, Coggi G, Cassani B, Bianchi P, Romagnoli S, Mandelli A, et al. Molecular changes in hepatocellular dysplastic nodules on microdissected liver biopsies. Hepatology 2000;32: 942 – 6. [60] Okuda T, Wakasa K, Kubo S, Hamada T, Fujita M, Enomoto T, et al. Clonal analysis of hepatocellular carcinoma and dysplastic nodule by methylation pattern of X-chromosome linked human androgen receptor gene. Cancer Lett 2001;164:91 – 6. [61] Aihara T, Noguchi S, Sasaki Y, Nakano H, Imaoka S. Clonal analysis of regenerative nodules in hepatitis C virus-induced liver cirrhosis. Gastroenterology 1994;107:1805 – 11. [62] Overturf K, al-Dhalimy M, Ou CN, Finegold M, Grompe M. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151: 1273 – 80. [63] Kojiro M. Pathology of hepatocellular carcinoma. In: Okuda K, Tabor E, editors. Liver cancer. New York: Churchill Livingstone; 1997. p. 165 – 87. [64] Kutami R, Nakashima Y, Nakashima O, Shiota K, Kojiro M. Pathomorphologic study on the mechanism of fatty change in small hepatocellular carcinoma of humans. J Hepatol 2000;33: 282 – 9. [65] Liver Cancer Study Group of Japan. The general rules for the clinical and pathological study of primary liver cancer [in Japanese]. 4th ed. Tokyo: Kanehara Co., Ltd; 2000. [66] Sakamoto M. Definition of early cancer and borderline lesions in the liver [in Japanese]. Pathol Clin Med 2001;19:93 – 7. [67] Kojiro M. Pathology of early HCC-progression from early to advanced. Hepatogastroenterology 1998;45:1203 – 5. [68] Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000;124:1599 – 607. [69] Falkowski O, West AB, Chiriboga L, et al. Intraseptal hepatocytes in cirrhosis: evidence for regeneration from stem cells. Mod Pathol 2001;14:216A.
Dysplastic nodules and hepatocarcinogenesis Neil D. Theise, MDa,*, Young Nyun Park, MDb, Masamichi Kojiro, MDc a
Department of Pathology, New York University Medical Center, Room 461, 560 First Avenue, New York, NY 10016, USA b Department of Pathology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea c Department of Pathology, Kurume University School of Medicine, Kurume, Japan
Recently, a growing literature has supported the concept that large nodules usually found in cirrhotic livers represent premalignant lesions in chronic liver disease. Using advanced imaging techniques in high-risk populations in Japan [1– 3], nodules suspicious for malignancy have often been identified and resected. Although some resected lesions were found to be small hepatocellular carcinomas (HCCs) [1,4,5], others were not. Some of these nonmalignant nodules were devoid of atypia; some had architectural or cytologic atypia insufficient for a diagnosis of HCC, although suggestive of a premalignant state; and others contained microscopic subnodules of HCC [1,4 – 7]. In follow-up studies from Japan, North America, Europe, and South Asia, including autopsy series [8– 10] and series of explants from liver transplant centers [11– 15], microscopic foci of HCC in the nodules were found and significant associations with HCC elsewhere in the same liver were established. These findings suggest that these nodular lesions are probably a frequent pathway of human hepatocarcinogenesis in a wide array of liver diseases and in diverse populations of patients. The emphasis in these early lesions has been somewhat different depending on the population of patients being studied. In Japan, where the bulk of available pathologic material comes from clinical biopsies and resections of lesions, more emphasis has been placed on higher-grade lesions and early HCCs, which are more likely to be detected radiologically and more closely associated with clinical progression, and therefore most important [14,16]. Outside of Japan, where large-scale screening of at-risk patients is not routine, most of the lesions have been identified and studied in the transplant setting, in whole cirrhotic livers. This source of pathologic material allows the physician to survey those lesions that
* Corresponding author. E-mail address: [email protected] (N.D. Theise). 1089-3261/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 3 2 6 1 ( 0 2 ) 0 0 0 0 6 - 5
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might not be detectable by conventional imaging but does not allow for follow-up data on the behavior of the lesions. Therefore, conclusions are based on associations, rather than observed progression.
Definitions and observations Dysplastic nodules consist of hepatocytes and almost always contain some intact portal tracts with a normal appearance. Because they are most often found in cirrhosis, they seem to correspond to what Edmondson called adenomatous hyperplasia (AH), although he viewed these lesions as having ‘‘limited growth potential’’ [17]. Other terms include macroregenerative nodule (MRN) [8,11, 12,15], dysplastic nodule [7], hepatocellular pseudotumor [13], and adenomatoid hyperplasia [14]. AH has been most widely used, though not exclusively, by researchers from Japan. MRN, named by Furuya et al [8] for the first Japanese autopsy study of the nodules, became the most widely accepted term in the earliest publications from North America and Europe. In 1997 a consensus document coauthored by investigators from Japan, North America, and Europe rejected all of these terms as being imprecise, if not actually misleading, and suggested the term dysplastic nodule, with further classification as low or high grade, to replace them [18]. Although researchers outside of Japan have generally accepted this new terminology, investigators in Japan have inconsistently adopted it, usually continuing to use the term AH. This review uses dysplastic nodule (DN) as the preferred term, recognizing that it, too, is problematic. DNs are defined grossly as large hepatic nodules that are distinct from the surrounding liver parenchyma in terms of size, color, texture, or the degree to
Fig. 1. Low-grade dysplastic nodule (DN). In this resection from a patient with primary biliary cirrhosis, a nodule distinct from surrounding nodules in size is easily seen; however, confirmation requires histologic examination.
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which they bulge from the cut surface of the liver (Fig. 1). A nodule is confirmed as a DN with histologic examination and the identification of intact portal structures distributed through the lesion. The number of these portal structures may be mildly or greatly reduced compared with a similar area of nondiseased hepatic parenchyma [19]. DNs have been found in a wide variety of chronic liver diseases [11,12,15], including processes that are hepatitic (hepatitis B, C, and autoimmune hepatitis), cholangitic (primary biliary cirrhosis, primary sclerosing cholangitis), metabolic (a-1-antitrypsin deficiency, primary hemochromatosis), and toxic (alcoholic liver injury). Typically, livers with DNs contain a small number of these nodules, rarely more than 10, although there are exceptions that are discussed later [8– 12,15]. DNs may be subclassified as low or high grade according to features listed in Table 1. Low-grade lesions may be devoid of atypia or display features of largecell change. They may be iron or copper retentive or they may be diffusely steatotic. They should not contain nodule-in-nodule – type lesions or small-cell change, or features closely associated with HCC; these changes would indicate a high-grade lesion. It is usually not possible to radiographically distinguish small HCCs from DNs or high-grade DNs from low-grade DNs with complete confidence [20,21], nor is it usually possible to reliably make such distinctions on the basis of gross morphology. Histologic examination, either by biopsy or examination of a resected specimen, is required for accurate classification.
Histologic features of low-grade DNs Low-grade DNs are well-defined nodules surrounded by a condensed rim of fibrous tissue similar to that surrounding cirrhotic nodules (Fig. 2). The nodules are therefore not truly encapsulated. Portal tracts, present in virtually all DNs, are most often uniformly distributed in low-grade DNs and may even be distributed in a virtually normal fashion with regularly intervening terminal hepatic venules. In
Table 1 Features that may be found in low-grade (ordinary) and high-grade (atypical) dysplastic nodules Low-grade DNs
High-grade DNs
Normal architecture Normal cytology or large-cell change Iron accumulation in otherwise nonsiderotic liver Iron resistance in otherwise siderotic liver Diffuse fatty change Copper accummulation Schirrous growth in the absence of other atypia
Focal pseudogland formation Diffuse small-cell change Nodule-in-nodule lesions with small-cell change Iron accumulation Iron resistance in siderotic nodule Fatty change Clear cell change Mallory body clustering Decreased reticulin fibers Features diagnostic of hepatocellular carcinoma
Abbreviation: DN, dysplastic nodule.
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Fig. 2. Low-grade dysplastic nodule. Microscopic examination of a distinctly large nodule (left of f ibrous septum) in hepatitis C cirrhosis confirms the absence of cytologic or architectural atypia (hematoxylin-eosin, original magnification 20).
some nodules, portal structures may be caught up in fibrous septa, which partially subdivide the nodule. The hepatocytes of low-grade DNs tend to be comparable in size to hepatocytes outside the lesion. The hepatocytes may display changes characteristic of the underlying liver disease affecting the surrounding liver, such as fatty change, Mallory bodies, or increased iron or copper deposition. These changes will be distributed in the DN as they are in surrounding cirrhotic nodules. Occasionally, a DN in a nonsiderotic liver may contain increased iron or a DN in an otherwise siderotic liver will be iron free; however, it would still be classified as low grade. Rarely, a similar increase in copper, in an otherwise copper-free liver, is also seen. These changes in DNs, when not confined to a subnodule within the lesion (ie, copper or iron retention and fat accumulation), may represent a marker of the clonality in the DN hepatocytes. Studies of large-cell change in cirrhosis and statistical evaluation of the association of this feature in DNs and HCCs [12,15] indicate the likelihood that large-cell change is usually a reactive, not a premalignant, change (although it also may serve as a marker for livers at increased risk for HCC). Therefore, it has been recommended that DNs containing large-cell change without other atypical features be classified as low-grade lesions [12]. Scirrhous changes, stellate fibrosis of DN portal tracts, or diffuse pericellular fibrosis in regions of DN parenchyma have previously been thought to be features of high-grade DNs; however, careful analysis of scirrhous change shows that it does not segregate with other features of high-grade lesions and appears in otherwise low- or high-grade lesions equally (Author’s unpublished data, N.D.T. 1999). Therefore, we would recommend that scirrhous change be excluded from the diagnostic criteria for high-grade DNs.
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Histologic features of high-grade DNs High-grade DNs are defined by the presence of small-cell change and/or architectural atypia. They are usually well circumscribed and surrounded by a condensed rim of fibrous tissue, as in low-grade lesions, although some may merge focally with adjacent liver parenchyma [22]. The atypical features in high-grade DNs may take a variety of forms and may be diffuse throughout the nodule or focal. Diffuse changes most often fall into the category of cellular atypia. The definition of cellular atypia in this setting should be limited to small-cell change: small, crowded hepatocytes with basophilic cytoplasm and an increased nuclear to cytoplasmic ratio [23]. Small-cell change appears to be consistently related to the development of HCC in a variety of studies and should remain a criterion for an atypical designation. This cellular feature is reported more frequently in studies from Japan than those from other countries [8– 12,15]; this discrepancy remains unexplained. Pseudoacinar structures resembling those seen in well-differentiated HCCs are a form of architectural atypia in high-grade DNs and may be either focal or diffuse. Focal atypia may merge with the surrounding DN parenchyma, but it more often occurs as a nodule-in-nodule lesion (Fig. 3) [12]. Such subnodules often appear to compress the adjacent DN parenchyma, and studies of the proliferation rates of the cells making up these lesions indicate that they are proliferating more rapidly than the surrounding tissue [24]. These subnodules may display small cell change (SCC) [15,23] but may also show changes that are not classically atypical, including fatty change [25], clear cell change [15,20], clusters of hepatocytes with Mallory’s hyaline [26], increased iron uptake within the DN [27], iron resistance in an otherwise siderotic nodule [27,28], and accumulation of copper-
Fig. 3. Small-cell change in a high-grade dysplastic nodule. Hepatocytes demonstrating small-cell change have hyperchromatic nuclei, nearly normal in size, with reduced, often basophilic, cytoplasm, thus an increased cytoplasmic ratio. Hepatocyte plates in small-cell change are often thickened or have a cobblestone appearance (upper right; hematoxylin-eosin, original magnification 40).
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Fig. 4. High-grade dysplastic nodule (DN) and hepatocellular carcinoma (HCC). DNs (open arrow) are often found in association with HCC (solid arrow) elsewhere in the liver. This patient with hepatitis B cirrhosis was found to have two nodules on imaging and underwent resection.
binding protein [29]. Some expansile subnodules do not display any distinctive cytologic features, although, architecturally, they may display a pseudoacinar growth pattern. Therefore, all subnodules, with or without distinctive cellular changes, are appropriately defined as architectural atypia on the basis of the expansile growth and should warrant classification of the entire DN as a highgrade lesion [12]. HCC may be identified in high-grade DNs (Fig. 4) [1,5,7 – 13,15,29 – 32]. These HCC microfoci may display any of the features seen in larger HCCs, although they are usually well differentiated. Typical growth patterns include pseudoacinus formation, thickened trabeculae, or a scirrhous growth. Common cytologic features include intracytoplasmic Mallory’s hyaline, fatty change, clear cell change, iron resistance, and multinucleation. Multiple foci of HCC may also be found in a single DN and the histologic features of these foci are often different from each other [30,32]. The DN parenchyma surrounding a microfocus of HCC will usually contain portal tracts and may consist of seemingly normal hepatocytes, suggesting a background of a low-grade MRN, or it may show atypia, indicating a high-grade background. Either way, by convention, DNs containing foci of HCC are classified as high grade.
The premalignant nature of DNs The association of DNs with HCC is demonstrated in two ways. First, as mentioned previously, DNs sometimes contain one or multiple microscopic foci of HCC. Second, DNs are sometimes found in livers that also contain grossly apparent HCC elsewhere (see Fig. 1). These relationships have been found to
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achieve statistical significance [11,12,15]. In addition to this statistical correlation, the atypical features often found in DNs include many that are thought to be premalignant. The clustering of hepatocytes containing Mallory’s hyaline [33,34] and foci of iron resistance in siderotic nodules [28,35] have been independently described as premalignant changes. Small cell change, considered premalignant on the basis of morphometric analysis, also has been seen and, in some series, is very common [5,10 –13,15,18]. Other features that are commonly identified in mature HCC are found in highgrade DNs. For example, immunohistochemical studies of DN sinusoids reveal increasing degrees of capillarization (ie, loss of endothelial fenestration, deposition of basement membrane, and expression of antigens such as factor VIII and CD34) with the development of atypia and HCC [36,37]. Studies of ploidy indicate an increased frequency of aneuploidy in high-grade DNs [38,39]. Immunohistochemical staining for a-fetoprotein [13,40,41] and p-Ras [13] demonstrates expression of these proteins in atypical foci and microfoci of HCC. Clonal stepwise progression has also been demonstrated in a subnodule of HCC arising in a DN by Tsuda et al [42]. Their study exploited integration of the hepatitis B surface antigen gene into host hepatocytes using restriction length polymorphism analysis, demonstrating that a microfocus of HCC consisted of cells derived from the same clone as the surrounding DN parenchyma. These findings represent static samplings of the process of malignant transformation. The association of DNs with HCC elsewhere in the same liver shows that DNs are at least a marker of a liver with a tendency to generate malignancies. Several studies, however, now demonstrate that, when followed over time by serial biopies or biopsy followed by radiographic changes indicative of expansile growth, there is progression from at least high-grade DN to HCC [4,5,43 –45]. In the previously described longitudinal studies, low-grade DNs are not as obviously premalignant or as indicative of neighboring malignant transformation as are high-grade DNs. The data addressing this difference appear contradictory. Studies of the various aspects of DNs mentioned—proliferation [13,40,46 – 48], p-Ras expression [13], ploidy [38,39], and endothelial and extracellular matrix alterations [36,37,49]—found similarities between high-grade DNs and HCC. In the absence of these similarities between low-grade DNs and HCC, most of these researchers concluded that high-grade lesions are related to HCC, whereas lowgrade lesions are related to regenerative nodules and are less clearly implicated in hepatocarcinogenesis. Data from the largest series of liver explants support and contradict this view of low-grade lesions [12]. A subset of livers was identified (13 of 155) that contained so many DNs as to be virtually uncountable. When large LCD was excluded as a criterion for atypia, the sampled DNs were low grade in all but one of these livers. None of these livers contained HCC. All such livers were in patients with chronic hepatitis (hepatitis B, hepatitis C, or autoimmune hepatitis), and the mean age of these patients was significantly lower than the other patients with few or no DNs. These findings suggest that these DNs are actually large regenerative nodules that have not yet scarred down into smaller nodules, and therefore examples of AH
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rather than neoplastic lesions. These DNs might also correlate with large cirrhotic nodules that became undetectable on long-term ultrasound surveillance in a study by Kondo et al [50]. However, statistical associations of low-grade lesions with coexistent HCC in this, as in earlier, series are strong, even when high-grade lesions are excluded from analysis [12,15]. Thus, the neoplastic, premalignant nature of low-grade lesions seems more open to question than it does for high-grade lesions, although perhaps most researchers (this study’s authors included) favor a distinction between these lesions. An explanation for the discrepancies, on the basis of hepatic stem cells, possibly resolves the apparent contradictions [51].
Speculations on the early stages of human hepatocarcinogenesis How DNs actually form has not yet been firmly established. One early view suggested that an ordinary regenerative nodule in cirrhosis becomes more rapidly proliferative, therefore larger. In turn, with the increased proliferation, it also becomes at greater risk for the carcinogenic ‘‘hits,’’ thereby giving rise to atypia and carcinoma [6]. Although simple, this hypothesis does not take into consideration three known facts about DNs. First, they can be found in livers in advance of cirrhosis and therefore do not always arise from a preexistent regenerative nodule [8,32,52]. Second, the presence of many intact portal tracts in most DNs, which have not yet been demonstrated to fully reconstitute after scarring and injury, suggests that they must preexist the formation of the DN, making it unlikely that a small cirrhotic nodule with few if any portal tracts could enlarge to a nodule with many portal tracts. Third, some DNs are clonal lesions, not hyperplastic phenomena [42,53 – 55]. To account for these features, studies have suggested an alternative process of DN development [32,51,56]. This alternate hypothesis has been able to predict some previously unexplored features of DNs and, perhaps, can be broadened to explain other types of borderline or early carcinomatous features. The steps of this hypothesis are as follows: 1.
2. 3.
4.
A clonal expansion of hepatocytes follows on the earliest carcinogenic events in response to any diffuse injury of the, liver which leads to increased hepatocyte turn over. These early hits lead to a clonal expansion of hepatocytes, which spread around adjacent portal structures rather than displacing them. As the rest of the liver becomes scarred, progressing to later stages of disease and eventually cirrhosis, the island of clonal hepatocytes, if resistent to the scarring affecting the rest of the liver, would remain intact— an island of relatively preserved hepatic parenchyma made up of neoplastic, clonal hepatocytes. With establishment of cirrhosis in the adjacent liver, the clonal expansion takes on the appearance of a large cirrhotic nodule.
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Having already undergone the earliest transforming events of hepatocarcinogenesis, the clonal, hepatocyte expansion remains at increased risk for later developments and thus the lesion becomes the likeliest site of full malignant transformation.
This hypothesis suggested a number of testable predictions that have resulted in supportive studies. First, expansion of a clonal population as described would not necessarily depend on rapid proliferation but might arise from a relatively low proliferation rate balanced by inhibition of apoptosis. In fact, many studies of proliferation have confirmed that the low-grade DNs and the background parenchyma of nodule-in-nodule – type high-grade DNs are no more proliferative, and are often less proliferative, than surrounding cirrhotic nodules [13,24,46 –48]. Of course, if these hepatocytes had a survival advantage compared with surrounding nonneoplastic hepatocytes, they would expand in a clustered fashion. One possibility is that they might be resistant to the disease damaging the surrounding parenchyma (eg, inability to be virally infected, absence of antigen presentation once infected, etc.) or they might have impaired mechanisms of apoptosis. We tested this latter possibility and found that apoptosis did appear diminished in low-grade DNs, only increasing with the emergence of atypia [57]. The rates of apoptosis in low-grade DNs were not significantly lower than those of surrounding regenerative nodules; however, the ratio of apoptosis to proliferation was increased in regenerative nodules, indicating that the hepatocyte populations, as expected, had a survival advantage when compared with non-DN hepatocytes. A key feature of our hypothesis of DN development is that the nodular appearance arises not from expansile growth, but because the clonal hepatocytes expand with reduced or absent scarring, while the surrounding, nonneoplastic liver becomes cirrhotic. Thus, the authors made a second testable prediction, which was confirmed: that there would be diminished stellate cell activation in DNs [58]. Activated stellate cells, shown by immunohistochemical staining for smooth muscle actin, were significantly fewer within DNs than in the surrounding cirrhotic nodules. To the authors’ knowledge, no other studies of this aspect of DN pathophysiology have been reported. Finally, clonality of many, if not all, DNs has been demonstrated in many studies [42,53 –55,59,60]. Many nodules that appear to regenerate have also been identified as clonal expansions, and neighboring, clustered, smaller cirrhotic nodules are often of the same clone [61]. This suggests, again, that the clonal expansion preceded the development of cirrhosis and that scarring subsequently subdivided the clonal expansion into separate nodules. Moreover, clonality of fields of hepatocytes has been identified in advance of cirrhosis, even in normal liver tissue [54]. Studies on the loss of heterozygosity have shown similar results [59].This suggests that some clonality is not related to neoplastic transformation but is a product of liver tissue formation in embryogenesis. However, these data confirm that clonal expansions can occur without displacing original portal structures, as predicted in the previously described model of DN development. How this precedes architecturally remains unclear. But, in animal models of
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Fig. 5. Hepatocellular carcinoma (HCC) in a dysplastic nodule (DN). (A) A nodule-in-nodule growth pattern (arrowhead) in a DN (hematoxylin-eosin, original magnification 10). (B) View of a region in Fig. 5A shows a moderately differentiated HCC in a background of otherwise unremarkable hepatocytes (hematoxylin-eosin, original magnification 40). (See also Color Plate 12.)
hepatocyte transplantation in which a small population of transplanted cells completely repopulates a genetically damaged liver, such as in the tyrosinemia mice of Markus Grompe et al [62], complete repopulation around native structures has been demonstrated.
Fig. 6. Multiple, small hepatocellular carcinomas (HCCs) in adjacent cirrhotic nodules in a patient with chronic hepatitis C; many of these nodules contain de novo HCC. The great variability of histologies in the HCCs strongly suggests that at least some of them are independent primaries, rather than invasion or metastasis into adjacent nodules by a single malignant clone. No HCC was identified in any other region of this explanted liver (Trichrome, original magnification 2). (See also Color Plate 13.)
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Toward a general concept of hepatocarcinogenesis Thorough, routine examination of cirrhotic liver explants inevitably will yield some early HCCs that do not appear to arise within a demonstrable DN. These are relatively infrequent and fall into two basic categories. The first is an HCC arising in a cirrhotic nodule that is not distinctive in any way, not differing from its neighbors in color, texture, bulging, or size (ie, what might be called a ‘‘de novo’’ HCC). This is easy to contain within our concept of DN, simply showing up our inability to distinguish neoplastic nodules unless they supply us with a clear morphologic marker of their distinctiveness. Such small cirrhotic nodules with HCCs might simply be small, low-grade DNs, without the size or accumulation of pigment to be noticable. The second category of small HCCs found incidentally in explants is rarer still. This category consists of multiple, very small HCCs, often with distinct morphologies and patterns of growth suggesting that they are independent lesions, arising in a discrete cluster of neighboring, small, cirrhotic nodules in a liver otherwise devoid of HCC (Fig. 5). Although no background DN is identified in these lesions, they likely represent the same process of HCC development from a prior clonal expansion as postulated for DN-related carcinogenesis. The clustering of so many nodules in a discrete area suggests that there is something distinctive about those clustered nodules. The comparison of this type of lesion to the clustered cirrhotic nodules of identical clones lends support to the idea that the clonality is the important feature, the appearance as a DN is merely an artifact of suppression of hepatic stellate cell (HSC) activation. Clonal expansions in which HSC activation occurs at normal levels in response to injury produce such clonal clusters and, just like DNs, these can give rise to HCC. We may therefore suggest a broader conception of hepatocarcinogenesis as portrayed in Fig. 7.
Fig. 7. Pathways of hepatocarcinogenesis. HSC = hepatic stellate cell; DN = dysplastic nodule; HCC = hepatocellular carcinoma.
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Areas of diagnostic uncertainty The generalized concept of early hepatocarcinogenesis can help to reconcile lesions, most often described by Japanese investigators, that do not match the classic descriptions of DNs. Lesions described as ‘‘early hepatocellular carcinoma’’ [63 –66], which often have indistinct borders or are identified in the absence of fully established cirrhosis, may simply reflect differing stages of scarring within the neoplastic expansions as opposed to the surrounding liver. The indistinct nature of the lesions suggests that they are a different neoplastic pathway; however, they may arise from a similar process (see Fig. 5). More difficult is the decision of where to draw the diagnostic line between dysplasia and carcinoma. An informal comparison of diagnoses for a range of hepatocellular neoplastic lesions among Japanese and non-Japanese pathologists (International Consensus Panel for Hepatocellular Carcinoma, Kurume, Japan, 2002, M.K., unpublished data) shows a lack of consistency within and between those two groups when it comes to deciding ‘‘cancer’’ versus ‘‘not yet cancer.’’ The type of lesion most difficult to diagnose is shown in Fig. 6. However, there is consensus among the reviewing pathologists on the more established carcinomas and low-grade lesions. Not surprisingly, then, the ‘‘borderline’’ category is the current area of controversy and leads to the greatest confusion when trying to compare studies from different investigators. Additional uncertainty in nodule characterization lies in the redundancy of so many adaptive or pathologic changes in the liver, with each of many morphologic changes arising from different causes. For example, fatty change in DNs or early HCC may arise from clonal changes in lipid metabolism or sensitivity to alcohol toxicity, or to changes in the ratio of arterial to portal blood flow, as the latter is lost during the neoplastic progression [64]. Mallory bodies may also represent a clonal marker of neoplastic alterations in gene expression, but could arise from chronic cholestasis in a nodule with incomplete biliary drainage, or an altered response to alcohol or lipid metabolism. Hepatocytes within portal tract or septal stroma may represent invasion [65 – 67], entrapment of nonneoplastic hepatocytes by scarring, ‘‘regression of cirrhosis’’ as described by Wanless et al [68], or hepatocyte regeneration from stem cells [69]. Thus, use of any of these histologic features for diagnostic purposes may be tremendously imprecise; each may indicate different things in different settings, such as evidence of clonality and neoplasia or evidence of simple regeneration.
Summary In the last decade, careful examination of explanted cirrhotic livers in liver transplant centers around the world has confirmed the findings of the earlier Japanese investigators: DNs (by this or any other name) represent hepatic, premalignant lesions in chronic liver disease. Careful examination of their gross and microscopic morphologies has led to the hypothesis of precirrhotic, spreading
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clonal expansions that are resistent to scarring, and that result in neoplastic islands of hepatic parenchyma. The resultant distinctive nodules, often marked by features suggestive of their clonality (such as increased pigment), are at increased risk for subsequent carcinomatous events, thereby giving rise to HCC. Specialized molecular and immunohistochemical studies confirm many aspects of this hypothesis. In suggesting that some aspects of DN pathophysiology are not integral to the carcinogenetic pathway (ie, inhibition of HSC inactivation), this hypothesis serves a broader purpose, explaining the various settings in which early HCCs are found in cirrhotic explants and in wedge resections of radiographically defined lesions. Discrepancies between Japanese and non-Japanese investigations regarding dysplasia and early HCCs reflect not different biologic pathways but differences in detection, interpretation, and application of nomenclature. These differences may fade away as more international collaborative work brings investigators of diverse nationalities into regular contact, supporting movement toward a commonly acceptable nomenclature and set of diagnostic criteria. Ultimately, an understanding of the pathophysiology of these lesions, through more detailed molecular and physiologic studies, should lead to more efficient and available early detection, and perhaps chemoprevention approaches to hepatic malignancy.
References [1] Arakawa M, Kage M, Sugihara S, Nakashima T, Suenaga M, Okuda K. Emergence of malignant lesions within an adenomatous hyperplastic nodule in a cirrhotic liver: observation in five cases. Gastroenterology 1986;91:198 – 208. [2] Okuda K. Early recognition of hepatocellular carcinoma. Hepatology 1986;9:751 – 5. [3] Shinagawa T, Ohto M, Kimura K, Tsunetomi S, Morita M, Saisho H, et al. Diagnosis and clinical features of small hepatocellular carcinoma with emphasis on the utility of real-time ultrasonography. A study in 51 patients. Gastroenterology 1984;86:495 – 502. [4] Kaji K, Terada T, Nakanuma Y. Frequent occurrence of hepatocellular carcinoma in cirrhotic livers after surgical resection of atypical adenomatous hyperplasia (borderline hepatocellular lesion): a follow-up study. Am J Gastroenterol 1994;89:903 – 8. [5] Takayama T, Makuuchi M, Hirohashi S, Sakamoto M, Okazaki N, Takayasu K, et al. Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 1990;336: 1150 – 3. [6] Sakamoto M, Hirohashi S, Shimosato Y. Early stages of multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol 1991;22:172 – 8. [7] Wada K, Kondo Y, Kondo F. Large regenerative nodules and dysplastic nodules in cirrhotic livers: a histopathologic study. Hepatology 1988;8:1684 – 8. [8] Furuya K, Nakamura M, Yamamoto Y, et al. Macroregenerative nodule of the liver. A clinicopathological study of 345 autopsy cases of chronic liver disease. Cancer 1988;61:99 – 105. [9] Terada T, Terasaki S, Nakanuma Y. A clinicopathologic study of adenomatous hyperplasia of the liver in 209 consecutive cirrhotic livers examined by autopsy. Cancer 1993;72:1551 – 6. [10] Vaiphei K, Ghosh UK, Vasishta RK. Incidence of adenomatous hyperplasia in postmortem cirrhotic livers and study of cellular proliferative indices by light microscopy. Indian J Gastroenterol 1999;18:7 – 10. [11] Ferrell L, Wright T, Lake J, et al. Incidence and diagnostic features of macroregenerative nodules vs small hepatocellular carcinoma in cirrhotic livers. Hepatology 1992;16:1372 – 81.
510
N.D. Theise et al. / Clin Liver Dis 6 (2002) 497–512
[12] Hytiroglou P, Theise ND, Schwartz M, Mor E, Miller C, Thung SN. Macroregenerative nodules in a series of adult cirrhotic liver explants: issues of classification and nomenclature. Hepatology 1995;21:703 – 8. [13] Le Bail B, Belleannee G, Bernard P-H, Saric J, Balabaud C, Bioulac-Sage P. Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 lesions in the cirrhotic explants of 10 adult French patients. Hum Pathol 1995;26:897 – 906. [14] Mion F, Grozel L, Boillot O, Paliard P, Berger F. Adult cirrhotic liver explants: precancerous lesions and undetected small hepatocellular carcinomas. Gastroenterology 1996;111:1587 – 92. [15] Theise ND, Schwartz M, Miller C, Thung SN. Macroregenerative nodules and hepatocellular carcinoma in forty-four sequential adult liver explants with cirrhosis. Hepatology 1992;16: 949 – 55. [16] Nakashima O, Sugihara S, Kage M, Kojiro M. Pathomorphologic characteristics of small hepatocellular carcinoma: a special reference to small hepatocellular carcinoma with indistinct margins. Hepatology 1995;22:101 – 5. [17] Craig JR, Peters RL, Edmondson HA. Tumours of the liver and intrahepatic bile ducts. Washington, DC: AFIP; 1989. [18] Wanless I, Callea R, Craig JR, et al. Terminology of nodular lesions of the liver: recommendations of the World Congress of Gastroenterology Working Group. Hepatology 1995;22: 983 – 93. [19] Nakashima Y, Nakashima O, Hsia CC, Kojiro M, Tabor E. Vascularization of small hepatocellular carcinomas: correlation with differentiation. Liver 1999;19:12 – 8. [20] Krinsky G, Theise ND, Rofsky N, Mizrachi H, Tepperman LW, Weinreb JC. Dysplastic nodules: arterial phase enhancement at CT and MR imaging with whole explant pathologic correlation—a case report. Radiology 1998;209:461 – 4. [21] Krinsky G, Lee VS, Theise N. Focal lesions in the cirrhotic liver—high resolution ex-vivo MR imaging with pathologic correlation. J Comput Assist Tomogr 2000;24:189 – 96. [22] Nakanuma Y, Terada T, Terasaki S, Ueda K, Nonomura A, Kawahara E, et al. ‘‘Atypical adenomatous hyperplasia’’ in liver cirrhosis: low grade hepatocellular carcinoma or borderline lesion? Histopathology 1990;17:27 – 36. [23] Watanabe S, Okita K, Harada T, Kodama T, Numa Y, Takemoto T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;55:2197 – 205. [24] Theise ND, Marcelin K, Goldfischer M, Hytiroglou P, Ferrell L, Thung SN. Low proliferative activity in macroregenerative nodules: evidence for an alternate hypothesis concerning human hepatocarcinogenesis. Liver 1996;16:134 – 9. [25] Terada T, Nakanuma Y, Hoso M, Saito K, Sasaki M, Nonomura A. Fatty macroregenerative nodule in non-steatotic liver cirrhosis: a morphologic study. Virchows Arch A Pathol Anat Histopathol 1989;415:131 – 6. [26] Terada T, Hoso M, Nakanuma Y. Mallory body clustering in adenomatous hyperplasia in human cirrhotic livers: report of four cases. Hum Pathol 1989;20:886 – 90. [27] Terada T, Nakanuma Y. Survey of iron-accumulative macroregenerative nodules in cirrhotic livers. Hepatology 1989;5:851 – 4. [28] Terada T, Nakanuma Y. Iron-negative foci in siderotic macroregenerative nodules in human cirrhotic livers: a marker of incipient pre-neoplastic or neoplastic lesions. Arch Pathol Lab Med 1989;113:916 – 20. [29] Nakanuma Y, Terada T, Ueda K, Terasaki S, Nonomura A, Matsui O. Adenomatous hyperplasia of the liver as a precancerous lesion. Liver 1993;13:1 – 9. [30] Ohno Y, Shiga J, Machinami R. A histopathological analysis of five cases of adenomatous hyperplasia containing minute hepatocellular carcinoma. Acta Pathol Jpn 1990;40:267 – 78. [31] Sakurai M, Wakasa K, Monden M, Yamada T, Kuroda C, Marukawa T, et al. Hepatocellular carcinoma in adenomatous hyperplasia of the liver. Cancer Chemother Pharmacol 1989; 23(Suppl):S110 – 3. [32] Theise ND, Lapook JD, Thung SN. A macroregenerative nodule containing multiple foci of hepatocellular carcinoma in a non-cirrhotic liver. Hepatology 1993;17:993 – 6.
N.D. Theise et al. / Clin Liver Dis 6 (2002) 497–512
511
[33] Nakanuma Y, Ohta G. Small hepatocellular carcinoma containing many Mallory bodies. Liver 1984;4:128 – 33. [34] Nakanuma Y, Ohta G. Is Mallory body formation a preneoplastic change? A study of 181 cases of liver bearing hepatocellular carcinoma and 82 cases of cirrhosis. Cancer 1984;55:2400 – 4. [35] Deugnier Y, Charalambous P, le Quilleuc D, Turlin B, Searle J, Brissot P, et al. Preneoplastic significance of hepatic iron-free foci in genetic hemochromatosis: a study of 185 patients. Hepatology 1993;18:1363 – 9. [36] Dhillon A, Colombari R, Savage K, Scheuer PJ. An immunohistochemical study of the blood vessels within primary hepatocellular tumors. Liver 1992;12:311 – 8. [37] Terada T, Nakanuma Y. Expression of ABH blood group antigens, receptors of Ulex europaeus agglutinin I, and factor VIII – related antigen on sinusoidal endothelial cells in adenomatous hyperplasia in human cirrhotic livers. Hum Pathol 1991;22:486 – 93. [38] Hoso M, Nakanuma Y. Cytophotometric DNA analysis of adenomatous hyperplasia in cirrhotic livers. Virchows Arch A 1991;418:401 – 4. [39] Orsatti G, Theise ND, Thung SN, Paronetto F. DNA image cytometric anlaysis of macroregenerative nodules (adenomatous hyperplasia) of the liver: evidence in support of their preneoplastic nature. Hepatology 1993;17:621 – 7. [40] Terasaki S, Terada T, Nakanuma Y, Nonomura A, Unoura M, Kobayashi K. Argyrophilic nucleolar organizer regions and alpha-fetoprotein in adenomatous hyperplasia in human cirrhotic livers. Am J Clin Pathol 1991;95:850 – 7. [41] Theise N, Fiel I, Hytiroglou P, Ferrell L, Schwartz M, Miller C, et al. Macroregenerative nodules in cirrhosis are not associated with elevated serum or stainable tissue alpha-fetoprotein. Liver 1994;15:30 – 4. [42] Tsuda H, Hirohashi S, Shimamoto Y, Terada M, Masegawa H. Clonal origin of atypical adenomatous hyperplasia and clonal identity with hepatocellular carcinoma. Gastroenterology 1998;95:1664 – 6. [43] Lencioni R, Caramella D, Bartolozzi C, Di Coscio G. Long-term follow-up study of adenomatous hyperplasia in liver cirrhosis. Ital J Gastroenterol 1994;26:163 – 8. [44] Sakamoto M, Hirohashi S. Natural history and prognosis of adenomatous hyperplasia and early hepatocellular carcinoma: multi-institutional analysis of 53 nodules followed up for more than 6 months and 141 patients with single early hepatocellular carcinoma treated by surgical resection or percutaneous ethanol injection. Jpn J Clin Oncol 1998;28:604 – 8. [45] Seki S, Sakaguchi H, Kitada T, Tamori A, Takeda T, Kawada N, et al. Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study. Clin Cancer Res 2000;6:3469 – 73. [46] Grigioni W, D’Errico A, Bacci F, Gaudio M, Mazziotti A, Gozzetti G, et al. Primary liver neoplasms: evaluation of proliferative index using MoAb Ki67. J Pathol 1989;158:23 – 9. [47] Terada T, Nakanuma Y. Cell proliferative activity of adenomatous hyperplasia in the liver and small hepatocellular carcinoma. An immunohistochemical study demonstrating proliferating cell nuclear antigen. Cancer 1992;70:591 – 8. [48] Tiniakos DG, Brunt EM. Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study. Liver 1999;19:58 – 68. [49] Park YN, Yang CP, Fernandez GJ, Cubukcu O, Thung SN, Theise ND. Neoangiogenesis and sinusoidal ‘‘capillarization’’ in dysplastic nodules of the liver. Am J Surg Pathol 1998;22:656 – 62. [50] Kondo F, Ebara M, Sugiura N, Wada K, Kita K, Hirooka N, et al. Histological features and clinical course of large regenerative nodules: evaluation of their precancerous potentiality. Hepatology 1990;12:592 – 8. [51] Theise N, Hytiroglou P, Thung S. Macroregenerative nodules and hepatocarcino-genesis. Il Friuli Medico Alpe Adria J Med 1994;49:637 – 50. [52] Matsuno Y, Hirohashi S, Furuya S, Sakamoto M, Mukai K, Shimosato Y. Heterogeneity of proliferative activity in nodule-in-nodule lesions of small hepatocellular carcinoma. Jpn J Cancer Res 1990;81:1137 – 40. [53] Aihara T, Noguchi S, Sasaki Y, Nakano H, Monden M, Imaoka S. Clonal analysis of precancerous lesion of hepatocellular carcinoma. Gastroenterology 1996;111:455 – 61.
512
N.D. Theise et al. / Clin Liver Dis 6 (2002) 497–512
[54] Ochiai T, Urata Y, Yamano T, Yamagishi H, Ashihara T. Clonal expansion in evolution of chronic hepatitis to hepatocellular carcinoma as seen at an X-chromosome locus. Hepatology 2000;31:615 – 21. [55] Paradis V, Laurendeau I, Vidaud M, Bedossa P. Clonal analysis of macronodules in cirrhosis. Hepatology 1998;28:953 – 8. [56] Theise ND. Macroregenerative (dysplastic) nodules and hepatocarcinogenesis: theoretical and clinical considerations. Semin Liver Dis 1995;15:360 – 71. [57] Park YN, Chae KJ, Kim YB, Park C, Theise ND. Increased activity of apoptosis and proliferation in human hepatocarcinogenesis. Cancer 2001;92:2733 – 8. [58] Park YN, Yang CP, Cubukcu O, Thung SN, Theise ND. Hepatic stellate cell activation in dysplastic nodules: evidence for an alternate hypothesis concerning human hepatocarcinogenesis. Liver 1997;17:271 – 4. [59] Maggioni M, Coggi G, Cassani B, Bianchi P, Romagnoli S, Mandelli A, et al. Molecular changes in hepatocellular dysplastic nodules on microdissected liver biopsies. Hepatology 2000;32: 942 – 6. [60] Okuda T, Wakasa K, Kubo S, Hamada T, Fujita M, Enomoto T, et al. Clonal analysis of hepatocellular carcinoma and dysplastic nodule by methylation pattern of X-chromosome linked human androgen receptor gene. Cancer Lett 2001;164:91 – 6. [61] Aihara T, Noguchi S, Sasaki Y, Nakano H, Imaoka S. Clonal analysis of regenerative nodules in hepatitis C virus-induced liver cirrhosis. Gastroenterology 1994;107:1805 – 11. [62] Overturf K, al-Dhalimy M, Ou CN, Finegold M, Grompe M. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151: 1273 – 80. [63] Kojiro M. Pathology of hepatocellular carcinoma. In: Okuda K, Tabor E, editors. Liver cancer. New York: Churchill Livingstone; 1997. p. 165 – 87. [64] Kutami R, Nakashima Y, Nakashima O, Shiota K, Kojiro M. Pathomorphologic study on the mechanism of fatty change in small hepatocellular carcinoma of humans. J Hepatol 2000;33: 282 – 9. [65] Liver Cancer Study Group of Japan. The general rules for the clinical and pathological study of primary liver cancer [in Japanese]. 4th ed. Tokyo: Kanehara Co., Ltd; 2000. [66] Sakamoto M. Definition of early cancer and borderline lesions in the liver [in Japanese]. Pathol Clin Med 2001;19:93 – 7. [67] Kojiro M. Pathology of early HCC-progression from early to advanced. Hepatogastroenterology 1998;45:1203 – 5. [68] Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000;124:1599 – 607. [69] Falkowski O, West AB, Chiriboga L, et al. Intraseptal hepatocytes in cirrhosis: evidence for regeneration from stem cells. Mod Pathol 2001;14:216A.