Space-occupying sizable nodules of the cirrhotic

Transcription

1 Hepatocellular Nodules in Cirrhosis: Focus on Diagnostic Criteria on Liver Biopsy. A Western Experience Massimo Roncalli The spectrum of so-called space-occupying small ( cm) sizable nodules arising in the cirrhotic liver includes a series of hyperplastic (large regenerative), dysplastic (low- and high-grade dysplastic), and malignant hepatocellular (well-differentiated hepatocellular carcinoma, HCC) nodules. Major progress in their classification and understanding was achieved through image analysis techniques and careful histological dissection of explanted native livers. Needless to say, the actual understanding of their natural history is crucial to a proper histological classification. The differential diagnosis of these hepatocellular nodules is difficult, particularly on biopsy specimens of focal liver lesions revealed by ultrasound (US), taken during the follow-up of cirrhotic patients. In this study we attempted to summarize, on the basis of our experience, essential clinicopathological features useful to distinguish the different nodules on needle biopsy. Synoptic tables of differential diagnosis and figures of elementar lesions, which have to be looked for, are provided. Only the continuous integration of clinical features, image analysis information of pathological findings, and follow-up data allows establishing the autonomy of these polymorphic and controversial entities and the boundaries between them. (Liver Transpl 2004;10:S9 S15.) Space-occupying sizable nodules of the cirrhotic liver include hyperplastic and neoplastic growths (benign, dysplastic, and malignant), whose nomenclature has been extensively revised. 1 Major progresses in their identification and understanding were provided by imaging evaluation in vivo, making possible the sampling of small early lesions (0.5 2 cm) and to follow their natural history Most of the advances in the field have been achieved through the systematic macro- and microscopical examination of native transplanted end-stage cirrhotic livers; however, there is a substantial lack of information on the morphological features of ultrasound (US)- detected hepatocellular nodules on liver biopsies. Indeed, the differential diagnosis of these lesions, while relatively easy on surgical specimens, is a challenging one on small liver biopsies, even for experienced hepatopathologists. The present work summarizes a Western experience in the field because it was originally promoted by the Italian Group of Gastrointestinal Pathology (GIPAD) with the aim of providing morphological criteria useful to classify liver biopsies on US-detected hepatocellular nodules raised in a cirrhotic, non-end-stage liver background. Clinical Information For a proper classification of hepatocellular nodules, the availability of the following clinical information is recommended: 1) Background liver: chronic liver disease / nonhepatitic, healthy liver. 2) Anamnestic data: contraceptive pills, drugs, dismetabolic disorders, hematologic diseases. 3) Biological markers: viral serology, oncofetal markers (AFP, CEA). 4) Vascular setting: portal hypertension; previous or ongoing portal thrombosis; vascular abnormalities. 5) Number, location, and size of nodule(s); when multiple, the sampled one should be properly identified. 6) Ultrasonographic pattern and its variation during follow-up. 7) Previous cyto/histopathological liver sampling and diagnostic report(s). Sampling An intra- and extralesional sampling is recommended. Subtle architectural and cytological abnormalities can Abbreviations: HCC, hepatocellular carcinoma; US, ultrasound; LRN, large regenerative nodule; LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule. From the Department of Pathology, University of Milan, Istituto Clinico Humanitas of Rozzano, Milan, Italy. Address reprint requests to Massimo Roncalli, MD, PhD, University of Milan Medical School, Department of Pathology, Istituto Clinico Humanitas, Via Manzoni 56, Rozzano, Italy. massimo.roncalli@unimi.it Copyright 2004 by the American Association for the Study of Liver Diseases Published online in Wiley InterScience ( DOI /lt Liver Transplantation, Vol 10, No 2, Suppl 1 (February), 2004: pp S9 S15 S9

2 S10 Roncalli Table 1. Diagnostic Approach to Hepatocellular Nodules In Cirrhotic Liver. Suggested Procedures As Related to Nodule Size Hepatocellular Nodules Imaging Techniques Sampling LRN LGDN HGDN HCC US sctscan NMR Angiography* FNAB i FNAB e FNA Size 1 cm 1 2 cm 2 3 cm 3 cm Abbreviations: LRN, large regenerative nodule; LGDN, low grade dysplastic nodule; HGDN, high grade dysplastic nodule; US, ultrasound; sctscan, spiral CT Scan; NMR, nuclear magnetic resonance; HCC, hepatocellular carcinoma. *Hepatic arteriography Lipiodol. Intralesional needlebiopsy (19 21 gauge). Extralesional needlebiopsy (19 21 gauge). Fine needle aspiration biopsy with Chiba needle (22 gauge).,, and indicate the coherence among the size & type of lesion, the sensitivity of imaging techniques and the way of sampling. be better recognized after a careful comparison with the extralesional referenced liver. The diagnostic contribution of imaging techniques and the suggested way of sampling are shown in Table 1. Sample adequacy is not only linked to the size of the biopsy, but is also dependent on the type of lesion, cell differentiation, liver background, as well as on the consistency between morphological and clinico-radiological features. Figure 1. Architectural elementar lesions in hepatocellular nodules. A: Plate thickening characterized by 2 3 cell-thick hepatocyte plates. B: Bulging clonal growth, clear cell type. Notice the compressed adjacent parenchyma (upper left). C: Map-like clonal growth, oncocytic cell type, clear cell type (D) and iron-free type (E) with indistinct cell boundaries. F: A hepatocyte pseudoglands (arrow) lacking Kupffer cell rimming at high and low magnification (G). H: Sinusoidal nuclear alignment. L: Nuclear crowding with clear cell changes as opposed to normal cell density (left). M: Unpaired arteries unassociated with portal tracts.

3 Hepatocellular Nodules in Cirrhosis S11 Figure 2. Cytological elementar lesions in hepatocellular nodules. A: Large cell changes in a periportal/periseptal. B: Small cell changes characterized by increased N/C ratio, cell density, and plate thickening. C: Hepatocyte tubularization near a focal lesion. The pericentral location and intraluminal bile plugs are secondary signs of liver compression. Pericentral liver cell anisocytosis is due to chronic cholestasis and should not be confused with large cell changes. Histological Elementar Lesions Architectural (recognizable at low magnification, ) Plate thickening (Fig 1A): hepatocyte plates between 2 adjacent sinusoids. In a normal condition, 1 cell thick, in regenerative and dysplastic conditions from 2 (usually) to 3 (rarely) cells thick and in malignant conditions 3 or more cells thick. Clonal growth (Fig. 1B E): a hepatocyte subpopulation with distinctive morphophenotypic features as compared to surroundings. This growth may be categorized into two groups: map-like and bulging. Bulging A hepatocyte nodular subpopulation with an expansive profile, sharp edges, and compression of the adjacent liver (Fig. 1B). Its growth fraction (as documented by PCNA and Mib1 immunostaining) may be appreciably increased as to surroundings. A sizable bulging lesion is the so-called nodule-in-nodule. Map-Like A hepatocyte nodular subpopulation recognizable at low magnification for homogeneous cytoplasmic features such as oncocytic (Fig. 1C) or clear (Fig. 1D) cells, fat/glycogen/iron/mallory body containing cells, ironfree cells (Fig. 1E), but with indistinct boundaries and without compression towards surroundings. 18 Pseudoglands or microacini (Fig. 1F and 1G): composed of tiny round hepatocyte aggregates with central pseudocanalicular lumen not rimmed by Kupffer cells. If not associated with chronic cholestasis, microacini are indicative of architectural disruption. Hepatocyte tubularization and regeneration, which occurs in a pericentral location following liver compression or chronic cholestasis, should not be confused with pseudoglands. Sinusoidal nuclear alignment (Fig. 1H): continuous hepatocyte nuclear alignment towards sinusoids. This feature, also, is indicative of an ongoing architectural disruption. Nuclear crowding (Fig. 1L): increased hepatocyte nuclear density documented at low magnification, as compared to surroundings. In doubtful cases, nuclear crowding is ascertained by semiquantitative analysis showing an increased cellularity, which should be at least twice as that seen in the surroundings. 15 Unpaired arteries (Fig. 1M): intraparenchymal arterioles unassociated to portal tracts. Cytological Large cell changes (ex large cell dysplasia) (Fig. 2A): groups of large periportal/periseptal hepatocytes with nuclear pleomorphism and hyperchromasia but usually preserved N/C ratio. Caution should be used in reporting large cell changes in the presence of chronic cholestasis and nuclear atypias have been reported in connection with chronic long-standing cholestasis. 19 Longstanding cholestasis can indeed induce a number of morphological hepatocyte abnormalities simulating architectural (nuclear crowding) and cytological (large and small cell changes) features that are characteristic of hepatocellular nodules (Fig. 2C). Small cell changes (ex small cell dysplasia) (Fig. 2B): foci of increased cellularity of monomorphous hepatocytes smaller than normal with increased N/C ratio. These changes are most frequently found in cirrhosis adjacent to hepatocellular carcinoma. Diagnostic Criteria General Features Hyperplastic and neoplastic hepatocellular nodules may be single or multiple. Size ranges from /3

4 S12 Roncalli Figure 3. Schematic representation of intra- (B) and extra- (A) lesional liver sampling for the detection of a hepatocellular nodule in cirrhosis. There are 2 possibilities: a) A B in terms of nodularity and cytoarchitectural features (left arrow). The sampling should not be considered adequate. b) A/B in terms of nodularity (right arrow). If a nodule greater than 0.8 (or far greater than adjacent nodules in B or to nodules detected in A) is depicted in B and does contain at least an unconnected portal tract, a diagnosis of hepatocellular nodule (HM) should be entertained. For further characterization of the nodules, see text. cm. Without an opposite proof, a lesion radiologically greater than 3 cm should be considered a hepatocellular carcinoma (HCC). Hepatocellular nodules include hyperplastic lesions such as large regenerative nodule (LRN) and neoplastic lesions such as low- and the highgrade dysplastic nodules. The ratio between hyperplastic and neoplastic nodules ranges from 4:1 to 10:1 and is about 4:1 between low- and high-grade dysplastic nodules ,20 Their prevalence in chronic HCV infection is variable according to different series (autopsy or posttransplanted native livers) ranging from 14 37%. Ultrasound-screened series showed a lower prevalence 17 ; in this setting, hepatocellular macronodules are mostly single. These findings can be explained by considering the natural history of native posttransplanted livers that is more advanced than that of livers of compensated cirrhotics undergoing a routine ambulatorial screening. While large regenerative nodules are thought to carry a malignant potential not greater than that of the adjacent cirrhosis, neoplastic nodules (low- and highgrade dysplastic) are considered precancerous. In clinical practice, their suggested treatment is a close follow-up for the former and percutaneous ethanol injection or thermoablation for the latter. HCC can develop within a given hepatocellular nodule or far from it Large cell changes detected in the cirrhotic surroundings of a hepatocellular macronodule are reported to statistically increase the risk of malignant transformation. 17 Hepatocellular macronodules are not associated with pathological AFP serum levels. Histopathology Hepatocellular nodules should contain unconnected portal tracts and centrolobular veins. The comparison between intra- and extralesional samples make their detection and classification more feasible (Fig. 3). Hepatocellular nodules should exceed at least twice the size of the Table 2. Hepatocellular Nodules ( cm) In Cirrhotic Liver. Key Diagnostic Features of Differential Diagnosis Cirrhosis LRN LGDN HGDN WDHCC Bulging clonal growth Map-like clonal growth Plate thickening Pseudoglands/microacini Unpaired arteries* Sinusoidal capillarization Nuclear peripheral alignment Cell crowding Large cell changes Small cell changes Reticulum framework Stromal/vascular invasion Abbreviations: LRN, large regenerative nodule; LGDN, low grade dysplastic nodule; HGDN, high grade dysplastic nodule; WDHCC, well differentiated hepatocellular carcinoma. Legend:, always;, maybe present;, absent. *Can be better visualized by Smooth Muscle Actin immunostaining. Can be visualized only by CD34 immunostaining.

5 Hepatocellular Nodules in Cirrhosis S13 surrounding cirrhotic nodules. At least 1 or 2 unconnected portal tracts are required to be detected within the putative lesion. In the case where both the intra- and the extralesional samples show overlapping, cirrhosis-like features, without a major nodule standing against the background, the sample should not be considered adequate for diagnosis (Fig. 3). Hematoxylin and eosin and a reticulum staining are very useful for a proper evaluation. Key morphological features of differential diagnosis 1,20 among nodules are detailed in Table 2. Large Regenerative Nodule (LRN) A hyperplastic lesion is usually recognized as a hepatocellular nodule at low magnification, standing without distinct cytoarchitectural features on the nodular surroundings. The intralesional sample may even resemble normal liver. The extralesional cirrhotic background, the finding of intralesional 2 cell-thick hepatocyte plates and the unconnected lesional portal tracts will contribute to elucidate the issue. 1,20 Low-Grade Dysplastic Nodule (LGDN) A LGDN is a clonal/neoplastic rather than a hyperplastic lesion, 21 although the issue is still under investigation. From a clinical point of view, both large regenerative and low-grade dysplastic nodules are lesions with putative low malignant potential. Low-grade dysplastic nodules are characterized by: 1) preserved hepatic architecture; 2) 2 cell-thick hepatocyte plates; 3) low-grade cytological atypias, generally in the form of large cell changes; 4) inconstant detection of map-like clonal growth; and 5) inconstant increase in the number of unpaired arterioles. High-Grade Dysplastic Nodule (HGDN) A HGDN is a neoplastic lesion with incipient malignancy. A parallel can be traced with gastrointestinal Figure 4. A: Intralesional sample of a high-grade dysplastic nodule showing a clonal growth of hepatocytes with clear cell changes and cell crowding (left). These changes are better appreciated if compared to the extralesional sample, which is devoid of these features (B). Figure 5. Progressive sinusoidal capillarization documented by CD34 immunostaining in intralesional samples of (A) a large regenerative nodule, (B) a low-grade dysplastic nodule, (C) a high-grade dysplastic nodule. D: Unpaired arteries documented in a case of high-grade dysplastic nodules by smooth muscle actin immunostaining. adenomas showing high-grade dysplasia (i.e., a neoplastic growth with high malignant potential). The main issue of differential diagnosis is well-differentiated HCC. A number of architectural abnormalities should be looked for and documented, such as 1) nuclear crowding; 2) 2 3 cell-thick hepatocyte plates; and 3) clonal growth such as map-like and the more ominous bulging (nodule-in-nodule) (Fig. 4A,B). Progressive sinusoidal capillarization can be detected by CD34 immunostaining (Fig. 5A C). 22,23 An increased number of unpaired arteries is usually detectable and may be confirmed by smooth muscle actin immunostaining (Fig. 5D). 22,23 Microacini are an important diagnostic feature (Fig. 1F and 1G). Distinction from well-differentiated HCC may be very difficult. 22 The proliferation rate as detected by Ki67 or PCNA immunostaining is usually increased, but not to the point to be semiquantitavely helpful in the differential diagnosis An essential feature for differential diagnosis is the uneven distribution of these pathological features in high-grade dysplastic as compared to a fully malignant nodule. A highgrade dysplastic nodule shows some features suggestive but not conclusive for a diagnosis of overt malignancy. Well-Differentiated HCC A diagnosis of well-differentiated HCC should be carefully considered when even architectural abnormalities (nuclear crowding, 3 or more cell-thick hepatocyte

6 S14 Roncalli Figure 6. Well-differentiated hepatocellular carcinoma: evenly thickened hepatocyte plates composed of monotous, medium-sized (A) to small (B) hepatocytes. The growth is only partially supported by the reticulin framework (C). D: A helpful clue for malignancy is the diffuse sinusoidal capillarization (CD34 immunostaining). plates) are associated with relative cell monomorphism (Fig. 6A,B). Nuclear density should be not less than twice the surroundings. A trabecular arrangement (which would facilitate the diagnosis) is not always detectable and the reticulum framework may be irregularly lost or reduced (Fig. 6C). Multiple microacinar structure with or without bile plugs, an abnormally high number of muscularized unpaired arterioles and of capillarized vessels (Fig. 6D), and infiltration of portal tract / fibrous septa / veins by single hepatocytes should be carefully looked for in the intralesional sample. 29 Intraparenchymal inflammatory cells and sinusoidal dilatation can also be detected. Like neoplastic nodules, well-differentiated HCC may contain subclonal populations with homogeneous cytoplasmic features (clear cells, small cells, etc.). The extensive lack of iron accumulation when the pigment can be demonstrated in the extralesional sample argues for a diagnosis of malignancy. Vascular invasion is diagnostic for malignancy, but this feature is barely detectable in small biopsies and an elastic staining may facilitate the search. 29 References 1. Terminology of nodular hepatocellular lesions. International Working Party. Hepatology 1995;22: Sadek AG, Mitchell DG, Siegelman ES, Outwater EK, Matteucci T, Hann HW. Early hepatocellular carcinoma that develops within macroregenerative nodules: growth rate depicted at serial MR imaging. Radiology 1995;195: Earls JP, Theise ND, Weinreb JC, DeCorato DR, Krinsky GA, Rofsky NM, et al. Dysplastic nodules and hepatocellular carcinoma: thin-section MR imaging of explanted cirrhotic livers with pathologic correlation. Radiology 1996;201: Borzio M, Borzio F, Croce A, Sala M, Salmi A, Leandro G, et al. Ultrasonography-detected macroregenerative nodules in cirrhosis: a prospective study. Gastroenterology 1997;112: Miller FH, Butler RS, Hoff FL, Fitzgerald SW, Nemcek AA Jr, Gore RM. Using triphasic helical CT to detect focal hepatic lesions in patients with neoplasms. AJR Am J Roentgenol 1998; 171: Terasaki S, Kaneko S, Kobayashi K, Nonomura A, Nakanuma Y. Histological features predicting malignant transformation of nonmalignant hepatocellular nodules: a prospective study. Gastroenterology 1998;115: Kanematsu M, Hoshi H, Yamada T, Murakami T, Kim T, Kato M, et al. Small hepatic nodules in cirrhosis: ultrasonographic, CT, and MR imaging findings. Abdom Imag 1999;24: Lim JH, Kim CK, Lee WJ, Park CK, Koh KC, Paik SW, et al. Detection of hepatocellular carcinomas and dysplastic nodules in cirrhotic livers: accuracy of helical CT in transplant patients. AJR Am J Roentgenol 2000;175: Kim CK, Lim JH, Lee WJ. Detection of hepatocellular carcinomas and dysplastic nodules in cirrhotic liver: accuracy of ultrasonography in transplant patients. J Ultrasound Med 2001;20: Krinsky GA, Lee VS, Theise ND, Weinreb JC, Rofsky NM, Diflo T, et al. Hepatocellular carcinoma and dysplastic nodules in patients with cirrhosis: prospective diagnosis with MR imaging and explantation correlation. Radiology 2001;219: Rode A, Bancel B, Douek P, Chevallier M, Vilgrain V, Picaud G, et al. Small nodule detection in cirrhotic livers: evaluation with US, spiral CT, and MRI and correlation with pathologic examination of explanted liver. J Comput Assist Tomogr 2001;25: de Ledinghen V, Laharie D, Lecesne R, Le Bail B, Winnock M, Bernard PH, et al. Detection of nodules in liver cirrhosis: spiral computed tomography or magnetic resonance imaging? A prospective study of 88 nodules in 34 patients. Eur J Gastroenterol Hepatol 2002;14: Martin J, Puig J, Darnell A, Donoso L. Magnetic resonance of focal liver lesions in hepatic cirrhosis and chronic hepatitis. Semin Ultrasound CT MR 2002;23: Denies L, Ernst O, Sergent-Baudson G, Duvet S, L hermine C. Contribution of spiral CT for the early diagnosis of hepatocellular carcinoma in cirrhotic patients. J Radiol 2002;83: Stoker J, Romijn MG, de Man RA, Brouwer JT, Weverling GJ, van Muiswinkel JM, et al. Prospective comparative study of spiral computer tomography and magnetic resonance imaging for detection of hepatocellular carcinoma. Gut 2002;51: Ward J, Robinson PJ. How to detect hepatocellular carcinoma in cirrhosis. Eur Radiol 2002;12: Borzio M, Fargion S, Borzio F, Fracanzani AL, Croce AM, Stroffolini T, et al. Impact of large regenerative, low grade and high

7 Hepatocellular Nodules in Cirrhosis S15 grade dysplastic nodules in hepatocellular carcinoma development. J Hepatol 2003;39: Shibata M, Morizane T, Uchida T, Yamagami T, Onozuka Y, Nakano M, et al. Irregular regeneration of hepatocytes and risk of hepatocellular carcinoma in chronic hepatitis and cirrhosis with hepatitis-c-virus infection. Lancet 1998;351: Natarajan S, Theise ND, Thung SN, Antonio L, Paronetto F, Hytiroglou P. Large-cell change of hepatocytes in cirrhosis may represent a reaction to prolonged cholestasis. Am J Surg Pathol 1997;21: Hytiroglou P, Theise ND. Differential diagnosis of hepatocellular nodular lesions. Semin Diagn Pathol 1998;15: Theise ND. Macroregenerative (dysplastic) nodules and hepatocarcinogenesis: theoretical and clinical considerations. Semin Liver Dis 1995;15: 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: Roncalli M, Roz E, Coggi G, Di Rocco MG, Bossi P, Minola E, et al. The vascular profile of regenerative and dysplastic nodules of the cirrhotic liver: implications for diagnosis and classification. Hepatology 1999;30: 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: Yamamoto K, Takenaka K, Kajiyama K, Shimada M, Shirabe K, Taketomi A, et al. Cell proliferation and cell loss in nodule-innodule hepatocellular carcinoma. Hepatogastroenterology 1999; 46: Faccioli S, Chieco P, Gramantieri L, Stecca BA, Bolondi L. Cytometric measurement of cell proliferation in echo-guided biopsies from focal lesions of the liver. Mod Pathol 1996;9: Terada T, Nakanuma Y. Cell proliferative activity in adenomatous hyperplasia of the liver and small hepatocellular carcinoma. An immunohistochemical study demonstrating proliferating cell nuclear antigen. Cancer 1992;70: Le Bail B, Belleannee G, Bernard PH, Saric J, Balabaud C, Bioulac-Sage P. Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 macronodular lesions in the cirrhotic explants of 10 adult French patients. Hum Pathol 1995;26: Nakano M, Saito A, Yamamoto M, Doi M, Takasaki K. Stromal and blood vessel wall invasion in well-differentiated hepatocellular carcinoma. Liver 1997;17:41 46.