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Liver cell dysplasia and early hepatocellular carcinoma
MINI-SYMPOSIUM: LIVER PATHOLOGY
Liver cell dysplasia and early hepatocellular carcinoma
increased in “low-risk” regions. The incidence in Hong Kong and Japan for example dropped by 35.1% and 42.7% respectively in the last two decades. This reduction is largely a result of Hepatitis virus B vaccination programs initiated in many East Asian and Asia-Pacific countries during the late 1980s.2 In contrast, the incidence has tripled in the United States between 1975 and 2005; similar rising trends are observed in the United Kingdom, Canada and Australia. Such growth is attributable to an epidemic of chronic hepatitis virus C infection, an increasingly young onset of alcohol consumption, and rising rates of obesity and diabetes mellitus with a consequent increase in prevalence of nonalcoholic steatohepatitis.3 Primary prevention through the control of risk factors contributing to the development of HCC is crucial for dealing with this challenging malignancy of global importance. Secondary prevention (i.e. interventions leading to the discovery and control of cancerous or precancerous processes while localized) is equally important to allow early diagnosis and prompt treatment of HCC to improve clinical outcome. Much of the high fatality rate of HCC (0.93 ratio of mortality to incidence) is due to the late detection of advanced HCC. Surveillance programs for at-risk populations have now been widely established.2,4,5 Advances in radiological techniques in such screening allow early detection of small hepatic nodules less than 2 cm in patients with chronic liver diseases. These small hepatic nodules represent a broad-spectrum of entities from benign, premalignant, to early malignant lesions. The accurate characterization of premalignant and early lesions of HCC poses a significant diagnostic challenge. The terminology for these lesions is based on a 1995 International Working Party (IWP) and is described in full below. Definition of the entities is important. The histological diagnosis of a macroscopically distinct small nodular lesion seen on imaging and on the cut surface of the subsequent resection specimens will be one of the following: large regenerative nodule, low-grade dysplastic nodule, highgrade dysplastic nodule, early HCC, progressed HCC (Table 1). There are other terms which refer to microscopic abnormalities that may be diagnostic features of the above; these include cytological changes in hepatocytes (large cell change, LCC; small cell change), and expansile foci of such cytological change (dysplastic foci). A histologically early HCC may have an indistinct margin macroscopically, and the term ‘vaguely nodular’ has been used for such lesions.
Anthony W H Chan Alastair D Burt
Abstract Hepatocellular carcinoma (HCC) is a major world health problem. Screening and diagnosis of early lesions of HCC allows prompt treatment to improve clinical outcomes. Early lesions of HCC include premalignant lesions and small HCCs. The former consists of hepatocellular cytological changes (large cell change, small cell change and iron-free foci), dysplastic foci and dysplastic nodules (low-grade and high-grade); while the latter is subdivided into early HCC and progressed HCC. There is significant overlap in pathological and radiological features between high-grade dysplastic nodules and early HCC making a precise diagnosis demanding even among experts. Various conventional and emerging potential diagnostic markers have been evaluated in assisting pathological diagnosis of early HCC. Translation of the concept of early HCC to daily clinical practice in term of diagnosis and tumour staging is challenging to clinicians, radiologists and pathologists.
Keywords dysplasia; hepatocellular carcinoma; liver; liver cancer
Introduction Hepatocellular carcinoma (HCC) is a major world health problem with a high incidence and fatality rate. Based on the latest data from GLOBOCAN by the International Agency for Research on Cancer, HCC is the fifth commonest cancer in men (523,000 cases, 7.9% of the total) and the seventh in women (226,000 cases, 6.5% of the total) worldwide.1 The majority (85%) of the cases occur in developing countries in Eastern and Southeast Asia, Middle and Western Africa. The annual age-standardized incidence rate in Eastern, South-Eastern Asia and Pacific regions is 21.8/100,000 with the highest rate in Mongolia (94.4/ 100,000), while it is much lower in North America (4.4/100,000) and the European Union (4.7/100,000). HCC leads to 477,000 and 217,000 annual deaths in men and women respectively around the World and is the third commonest cause of cancer death; the geographical distribution of the mortality matches that observed for incidence. The epidemiology of HCC has changed over recent decades. The incidence of HCC has decreased in “high-risk” regions but
Premalignant lesions in human hepatocarcinogenesis The development of HCC (hepatocarcinogenesis) is a multistep process characterized by progressive accumulation and interplay of genetic and epigenetic changes leading to uncontrolled clonal proliferation, local invasion and distant metastasis. During the process, a number of phenotypic changes arise, often designated as premalignant lesions.
Anthony W H Chan FRCPA is at the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. Conflicts of interest: none.
Microscopic changes, including dysplastic foci Large cell change, formerly known as large cell dysplasia, was first described by Anthony et al. and initially referred to “liver cell dysplasia” in 1973.6 It is characterized by a group of hepatocytes with cellular and nuclear enlargement, nuclear pleomorphism and multinucleation, prominent nucleoli, and
Alastair D Burt FRCPath is Professor at the Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. Conflicts of interest: none.
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Summary of pathological and radiological features of small nodular lesions in the cirrhotic liver15,16 LRN
Pathological appearance (gross) Pathological appearance (microscopic) Nodule-in-nodule Cell crowding, compared with background cellularity Clone-like population Thickened trabeculae Pseudoglands Deficiency of reticulin fibres Intranodular portal tract Unpaired arteriole Stromal invasion Vascular invasion Nuclear atypia Radiological appearance Arterial supply Isovascular/ hypovascular Portal venous supply
þ
LGDN
HGDN
Early HCC (WD-HCC)
Progressed HCC (MD-HCC)
Usually distinctly nodular
Usually vaguely nodular
Vaguely nodular
Distinctly nodular
(<1.5)
þ (1.5e2)
þ (>2)
þ (>2)
(2 cells thick)
þ (2e3 cells thick) þ
þ (3 cells thick) þ
þ (3 cells thick) þ
þ
þ þ
þ þ þ
þ þ
Isovascular/ hypovascular
Isovascular/ hypovascular
Hypervascular
þ
þ
Isovascular/ hypovascular/rarely hypervascular þ
LRN, large regenerative nodule; LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule; HCC, hepatocellular carcinoma; WD, well-differentiated; MD, moderately differentiated; , absent; , may be present but not necessarily detected in core biopsy; þ, usually present and detected in core biopsy.
Table 1
LCC compared to adjacent HCC.7,8 It is currently believed that LCC usually represents the pathophysiological nuclear polypoidy which is a degenerative senescent change as a consequence of chronic liver injury. In this respect, LCC is best regarded as a predictive marker associated with HCC rather than a genuine premalignant lesion. However, recent data suggests that LCC may, in some situations, be a genuine premalignant lesion.9 Small cell change was initially described by Watanabe et al. as “liver cell dysplasia of small-cell type” in 1983.10 It is characterized by a collection of hepatocytes with a decreased cell volume, minimal nuclear pleomorphism, an increased N:C ratio, and increased nuclear density (Figure 2). It is generally believed to be an early premalignant lesion because of a higher prevalence in cirrhotic livers with HCC than in those without, a high proliferative activity, a morphological continuum between small cell change and HCC, a similar immunophenotype to hepatic progenitor cells, and similar chromosomal alterations in small cell change and adjacent HCC. Plentz et al. demonstrated that markedly reduced expression of p16 and p21 coincided with further telomere shortening and an accumulation of DNA damage in small cell change and HCC compared to LCC and cirrhotic nodules suggesting that small cell change represents a more “advanced” lesion than LCC.11 Iron-free foci in genetic haemochromatosis were shown to be more frequent in livers with HCC (50.0%) than those without (8.3%), and demonstrated a high proliferative index and co-existing LCC/small cell change (71.4%).12 HCCs in genetic
preserved nucleocytoplasmic (N:C) ratio (Figure 1). It was initially considered a premalignant lesion because of a frequent association with HCC and the presence of an abnormal and increased DNA content. However, this concept was challenged by observations of a normal N:C ratio, low proliferative index, high apoptotic activity, lack of a direct transition from LCC to carcinoma, and the absence of relevant genetic abnormalities in
Figure 1 Large cell change featured by cellular and nuclear enlargement, nuclear pleomorphism and multinucleation, prominent nucleoli, and preserved N:C ratio (H&E, original magnification 400).
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Figure 2 Small cell change featured by decreased cell volume, minimal nuclear pleomorphism, an increased N:C ratio, and increased nuclear density (H&E, original magnification 200).
Figure 3 Oncocytic change featured by abundant eosinophilic granular cytoplasm (H&E, original magnification 200).
increased cell density in HGDN can be sometimes up to twice that of the surrounding liver. Both also often exhibit features suggestive of a clonal origin, such as iron or copper accumulation, or steatosis in the context of a background liver without significant fatty change. LGDN is usually distinct rather than vaguely nodular due to the presence of a condensed fibrous scar or pseudocapsule at its periphery. Architectural atypia is absent, while cytological atypia is minimal with occasional LCC and absent mitoses. It often contains intranodular portal tracts, with only very rare aberrant unpaired or non-triad arterioles. Its blood supply is entirely derived from portal veins, and hence it is either hypovascular or isovascular in the arterial phase of contrast-enhanced imaging. It is impossible to confidently distinguish between LGDN and LRN by morphology alone in the current consensus and it has been argued that the latter term should be dropped. HGDN is usually vaguely nodular and contains significant architectural and/or cytological atypia. Atypical architectural features include nodule-in-nodule pattern (Figure 4), pseudoglandular or acinar formation (Figure 5), and thickened trabeculae up to three cells thick. Cytological changes comprise focal or diffuse small cell change, iron-free foci, focal steatosis with Mallory-Denk bodies (Figure 6) or p62 inclusions (Figure 7), and clear cell change. Among all these atypical changes a nodule-in-nodule pattern is the most worrying feature because HCC not uncommonly emerges from such “sub-nodules”. HGDN contains fewer intranodular portal tracts and more aberrant unpaired arterioles than LGDN. Its main blood supply is still derived from the portal vein and hence it is either hypovascular or isovascular on imaging.
haemochromatosis are typically iron-free, and the sequential development from iron-free nodules to iron-free HCC in a recent rat model further support that iron-free foci are premalignant lesions.13 However, the underlying mechanisms of iron resistance in these foci and subsequent progression into HCC remain unknown. A dysplastic focus was defined by the IWP as a microscopic abnormality less than 1 mm in diameter, consisting of an expansile group of hepatocytes with cytological features suggestive of premalignant change, particularly small cell change and iron-free foci. Apart from these well-documented atypias, there are other hepatocellular cytological changes, collectively termed “foci of altered hepatocytes (FAH)”, described in several animal models in early hepatocarcinogenesis caused by chemicals, radiation, and chronic infection with hepadnaviruses.14 Although some FAH, including glycogen-storing clear cell foci, clear cellpredominated mixed cell foci and oncocytic foci (Figure 3), have been also observed in chronic liver disease with or without HCC in humans, there is insufficient evidence to establish the definite role of FAH in human hepatocarcinogenesis. Large regenerative nodules, and dysplastic nodules In the 1995 paper published by the International Working Party (IWP) of the World Congresses of Gastroenterology the following consensus nomenclature and morphological criteria for hepatocellular nodular lesions was proposed15: A large regenerative nodule (LRN) (or macroregenerative nodule) was seen as a nodule with a minimum diameter of 5 mm, and one which is more distinctive and larger than surrounding cirrhotic regenerative nodules. It is composed of hepatocytes that are cytologically bland without any architectural or cytological atypia. It is commonly situated adjacent to a large portal tract. A dysplastic nodule (DN) was defined as a nodule macroscopically distinct from surrounding cirrhotic regenerative nodules due to its size (varied from few millimetres to few centimetres, mostly less than 15 mm), colour, texture, and degree of bulging. It is further classified into low-grade (LGDN) and highgrade (HGDN) according to the degree of architectural and/or cytological atypia. Both LGDN and HGDN are hypercellular;
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Small HCC, early HCC and progressed HCC Small HCC was defined by the IWP as an HCC less than 2 cm, and subdivided into two groups, early HCC and progressed HCC.16,17 Early HCC is also known as “small HCC of vaguely nodular type” and “small HCC with indistinct margin”. Pathologically, this is a well-differentiated HCC with vague nodulation, indistinct borders and various combinations of (1) increased cell density more than twice that of the surrounding liver; (2) small cell change; (3) pseudoglandular pattern; (4) irregular trabecular
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Figure 4 Nodule-in-nodule pattern in a high-grade dysplastic nodule (Reticulin, original magnification 25).
Figure 6 Mallory-Denk bodies. Ropey deeply eosinophilic cytoplasmic inclusions (H&E, original magnification 200).
Progressed HCC is also termed “small HCC of distinct nodular type” and “small nodular HCC with distinct margins”. Pathologically, it is a moderately differentiated HCC with a distinctly nodular appearance often with a fibrous pseudocapsule. It resembles classical HCC with typical trabecular and pseudoglandular architectures, numerous aberrant unpaired arterioles, and absence of intratumoural portal tracts. Vascular invasion and intrahepatic metastasis are found in 29% and 21% of cases respectively.17 Radiologically, progressed HCC is hypervascular as it derives blood exclusively from the arterial supply as classical HCC does. In addition to differences in the pathological and radiological appearances between early HCC and progressed HCC, the latter shows a less favourable outcome than early HCC with 5year overall survivals of 54% and 93% respectively.17
pattern with occasional more than three cells thick; (5) diffuse fatty change; (6) intratumoural portal tracts; and (7) unpaired arterioles. Vascular invasion and intrahepatic metastasis are absent or exceptionally rare. Radiologically, early HCC is hypovascular, isovascular, or very rarely hypervascular. Marked overlapping in pathological and radiological features between early HCC and HGDN makes a precise diagnosis demanding. The key pathological feature for distinguishing early HCC from HGDN is the presence of stromal invasion, which is defined as infiltration of tumour cells into intratumoural portal tracts or fibrous septa (Figure 8). When stromal invasion is difficult to establish unequivocally on routine histology, the absence of significant ductular reaction (with the aid of immunostaining of cytokeratins 7/19) favours genuine stromal invasion rather than pseudoinvasion.18 Another histological investigation that may be helpful is the reticulin stain; convincing deficiency of a reticulin framework in the liver cell plates is a useful diagnostic feature of HCC, but this is not necessarily deficient, and so a normalappearing reticulin does not exclude early HCC.
Updated international consensus and discrepancy between east and west In order to achieve a refined and updated international consensus of hepatic nodules, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in 2002
Figure 5 Pseudoglandular pattern in a high-grade dysplastic nodule (H&E, original magnification 100).
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Figure 7 p62 inclusions. Large oval-shaped deeply eosinophilic cytoplasmic inclusions (H&E, original magnification 200).
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for AFP. Although AFP is a highly specific (95e100%) tissue marker, it is not an optimal diagnostic marker because of low sensitivity (25e30%) and a heterogeneous patchy staining pattern. Its application in early HCC detection is further hindered as Fujioka et al. found that none of 13 cases of early HCC were immunoreactive for AFP.20 AFP can be fractionated by affinity electrophoresis into three glycoforms: L1, L2, and L3 based on the reactivity with the lectin Lens culinaris agglutinin. AFP-L1 is the major glycoform in serum of patients with chronic hepatitis and cirrhosis, while AFPL2 and AFP-L3 are glycoforms more specifically produced by yolk sac tumours and HCC respectively. The percentage of AFPL3 over total AFP level (AFP-L3%) is not only a highly specific (>93%) serum marker for HCC, but also an adverse prognostic marker associated with vascular invasion and intrahepatic metastasis.19,21 Although the detection rate of small HCC by AFPL3% is low (22.2e23.0%), there is a potential value for differentiation between early HCC and progressed HCC.19,22 Kumada et al. found that none of 15 cases (0%) with early HCC and 14 of 45 cases (31.1%) with progressed HCC showed diagnostic levels of AFP-L3%.22 An antibody against AFP-L3 for immunohistochemistry however is not yet commercially available. Des-gamma-carboxy prothrombin (DCP), also known as protein induced by vitamin K absence or antagonist-II (PIVKAII), is an abnormal prothrombin produced by HCC tumour cells. It has been used as a specific serum marker, as well as a poor prognostic marker because of its association with early portal vein invasion, intrahepatic metastasis and capsular infiltration. Compared to serum AFP in the detection of small HCC, it offers comparable sensitivity (44e54%) and higher specificity (95e 99%).21 Few studies have investigated tissue DCP expression by immunohistochemistry; these demonstrated unsatisfactory sensitivities of 63.2% (50.0e81.8%), 48.1% (29.6e75%) and 47.8% (39.3e61.0%) in identifying all, well-differentiated and small HCCs respectively.20,23,24 Miskad et al. found that eight of 12 early HCCs (66.7%) and none of nine DNs (0%) were immunoreactive for DCP, which could be used as a potential diagnostic aid.24 CD34 is an intercellular adhesion glycoprotein and one of the commonly used vascular endothelial markers. Sinusoidal endothelium in normal liver is devoid of CD34 immunoreactivity. However, many non-neoplastic and neoplastic liver diseases lead to distortion of the normal hepatic microcirculation and the formation of aberrant new vessels (neo-vascularization), resulting in sinusoidal capillarization. Two distinct patterns of capillarization can be demonstrated by immunostaining of CD34 e complete and incomplete patterns. The former is characterized by a diffuse sinusoidal expression of CD34 within the entire lesion, while an incomplete pattern is featured by focal and preferentially peripheral sinusoidal expression of CD34 in the lesion. The complete CD34 staining pattern is sensitive in detecting 93.2% (82.5e100%) and 89.8% (82.7e100%) of all and well-differentiated HCCs respectively with a high specificity of 96.2% (82.5e100%).25e28 It is also observed in 3.7% of FNH and 20.7% of HCA but this does not create any major diagnostic issues when tackling a nodular lesion in a cirrhotic liver together with radiological correlation. Cirrhotic nodules, LRN and LGDN do not exhibit the complete pattern. With respect early HCC and its major mimicker HGDN, complete CD34 staining is observed in
Figure 8 Stromal invasion in an early HCC. Infiltration of tumour cells into intratumoural portal tracts without accompanied ductular reaction (H&E, original magnification 100).
with several subsequent meetings until 2007.16 The latest ICGHN comprised of 34 hepatopathologists and two clinicians from 13 countries, including most members of the original IWP who are still active and all the participants from the first ICGHN meeting. A total of 26 surgically resected nodules from 23 patients with chronic hepatitis B or C were selected from Japanese and Korean medical centres; all the nodules were less than 2 cm. For each case, all pathologists in the study group independently reviewed brief clinical data, a gross picture, and an H&E slide containing the entire width of the lesion, and classified the lesion according to the IWP criteria. There was little disparity in distinguishing early HCC/HGDN from LGDN/LRN or progressed HCC. However, the differentiation between early HCC and HGDN showed a significant disagreement between Eastern and Western pathologists. The former had much higher tendency to diagnose early HCC. At the first conference, the diagnosis of early HCC and HGDN showed wide inter-observer variation with very low Kappa value of 0.30. By the addition of clearly defined stromal invasion to the diagnostic criteria for early HCC the discrepancy decreased and the Kappa value rose to 0.49 at the subsequent conference. However, the inter-observer agreement is still suboptimal and further fine-tuning of the diagnostic criteria and exploring suitable diagnostic markers are necessary.
Early HCC and conventional diagnostic markers There are several well-established serum and tissue markers for HCC. Alpha-fetoprotein (AFP) is a major plasma glycoprotein produced by the yolk sac and liver during normal foetal development. It has been used as a serum marker for HCC since the 1970s. However, serum AFP as a diagnostic/screening test for small HCC is of limited value. Serum AFP has a sensitivity of only 49e51% and a specificity of 83e86% in the detection of small HCC.19 Tissue AFP expression demonstrated by immunohistochemistry in a hepatic lesion is diagnostic of malignant hepatocellular neoplasm (HCC or hepatoblastoma) excluding rare metastatic yolk sac tumour or metastatic hepatoid carcinoma. Other HCC mimickers including LRN, DN, focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are all negative
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69.0% and 2.0% respectively, and therefore this could serve as a useful diagnostic tool.27,29
Glutamine synthetase (GS) catalyzes the synthesis of glutamine, the major energy source of tumour cells. It plays an important role in ammonia detoxification, nitrogen homeostasis, and acidebase balance in the mammalian liver. Christa et al. showed up-regulation of GS mRNA, protein, and enzyme activity in HCC, and subsequently Cadoret et al. observed the activation of Wnt/beta-catenin pathway leading to this overexpression.38,39 Osada et al. demonstrated a progressive increase in GS immunoreactivity from DN, early HCC to advanced HCC, suggesting an involvement in early hepatocarcinogenesis.40 A recent proteomic analysis of HCC less than 3 cm consolidated the role of GS in early hepatocarcinogenesis.41 Immunoreactivity of GS is observed in 44.8% and 20.8% of all and early HCCs respectively, compared with 4.1%, 0% and 0% of HGDN, LGDN and cirrhosis respectively. Similarly to HSP70, its application in differentiation between early HCC and HGDN is limited by low sensitivity and accuracy.32,40 A panel of three markers, GPC3, GS and HSP-70 is currently emerging as a helpful strategy for diagnosing HCC. Positivity for any two of the three markers was found to indicate malignancy with 72% sensitivity and 100% specificity.42 When the same panel was used to retrospectively distinguish between HCC and HGDN on liver biopsies, the sensitivity decreased to 49% while the specificity remained 100%.32 A recent study suggested that the addition of clathrin heavy chain (CHC) to the existing panel of three markers produced an improvement in the sensitivity and diagnostic accuracy when two markers were positive whilst still retaining the 100% specificity.43
Early HCC and emerging potential diagnostic markers With a better understanding of genetic and epigenetic changes in early HCC through different high throughput genome-wide and other novel emerging molecular methodologies, we may have a better insight of early hepatocarcinogenesis and the identification of potential diagnostic markers for detection of early HCC. Heat shock protein 70 (HSP70) is a family of proteins implicated in carcinogenesis, regulation of cell-cycle progression and apoptosis. Many HCCs arise from liver diseases with chronic necroinflammation and fibrosis, which induce the synthesis of such heat shock proteins. Chuma et al. employed oligonucleotide arrays of 12,600 genes and demonstrated that HSP70 is the most abundantly up-regulated gene in early HCC. This study also found a progressive increase in messenger RNA (mRNA) levels and tissue expression of HSP70 from LGDN, HGDN, early HCC to progressed HCC by using quantitative realtime reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry respectively.30 Subsequent studies by immunohistochemistry from other groups confirmed this finding, showing overexpression of HSP70 in 58.7%, 46.7% and 22.7% of all, well-differentiated and early HCCs respectively, in contrast to 9.2%, 0% and 0% of HGDN, LGDN and cirrhosis respectively.31e33 However, HSP70 immunohistochemistry, its application in differentiation between early HCC and HGDN is restricted by its low sensitivity and positive predictive value.31,32 Cyclase-associated protein 2 (CAP2) is a protein involved in the interaction between adenylyl cyclase and actin but its exact function in human remains unknown. In the same genome-wide study of early HCC of Chuma et al., CAP2 was another upregulated gene.30 The follow-up study by the same group demonstrated through qRT-PCR and immunohistochemistry stepwise increments in CAP2 mRNA and protein levels from DN, early HCC to progressed HCC.34 Although this is a promising diagnostic marker for early HCC, there are no other studies yet available to validate the findings. Glypican-3 (GPC3) is a member of glycosyl-phosphatidylinositol anchored heparin sulphate proteoglycans. It is normally expressed in foetal but not adult liver. It promotes the growth of HCC tumour cells by activating Wnt/beta-catenin and insulin-like growth factor signalling pathways.35,36 Okabe et al. identified GPC3 being one of 86 up-regulated genes in HCC by using complementary DNA microarrays consisting of 23,040 genes.37 Llovet et al. further demonstrated GPC3 to be one of five upregulated genes in early HCC compared to DN by analyzing transcriptional profiles of 55 candidate genes through qRT-PCR, and showed tissue expression of GPC3 by immunohistochemistry in HCC but not DN.35 Several immunohistochemical studies illustrated that GPC3 is sensitive in detecting 78.6% (68.1e88.8%) and 68.8% (50.0e94.4%) of all and welldifferentiated HCCs respectively with the overall specificity of 92.9% (87.0e98.2%).27,32,33 It is of potential diagnostic use because of a significantly different expression in early HCC (80.0%) compared with HGDN (15.5%).27,32
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Clinical implications of early HCC Diagnosis: the apparently favourable prognosis of early HCC emphasizes its importance in incorporation into clinical practice. Ultrasonography (USG) is the radiological test for surveillance for HCC in cirrhotic patients recommended by the European Association for the Study of the Liver (EASL), the American Association for the Study of Liver Diseases (AASLD) and the AsianPacific Association for the Study of the Liver (APASL).2,4,5 However, the USG detection rate drops significantly from 65 e80% of conventional HCC to 21% of small HCC.5,44 To establish a definitive radiological diagnosis of small HCC, other modalities including contrast-enhanced USG and magnetic resonance imaging remain suboptimal with unsatisfactory sensitivity of 61.7% and 57.1% respectively.45 The major underlying hindrance is the hypovascularity of early HCC. AASLD recommends liver biopsy for hepatic nodules between 1 and 2 cm without a characteristic radiological vascular pattern of HCC.5 However, a confident histopathological distinction between early HCC and HGDN is often impossible because intranodular portal tracts or fibrotic septa (essential for the determination of stromal invasion) may not be present in needle biopsy specimens. Distinguishing LRN/LGDN and progressed HCC arising in pre-existing DN from HGDN/early HCC is sometimes not straightforward because of lesional heterogeneity and sampling error. Nevertheless, liver biopsy remains more sensitive than imaging studies for the detection of early HCC.45 The application of immunohistochemical markers may be helpful, but one should remember that the accuracy of these markers for diagnosis of early HCC in biopsy specimens is not properly validated.
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Diagnostic accuracy of immunohistochemical tumour markers in differentiation between high-grade dysplastic nodule and early hepatocellular carcinoma Tumour marker
HGDN
DCP24 CD34 (complete capillarization)27,29 HSP7031,32 GPC327,32 GS32,40 Any one of HSP70/GPC3/GS32 Any two of HSP70/GPC3/GS32 Any one of HSP/GPC/GS/CHC43 Any two of HSP/GPC/GS/CHC43
0% 2.0% 9.2% 15.5% 4.1% 22.0% 0% 18.8% 0%
(0/9) (2/98) (9/98) (15/97) (3/73) (11/50) (0/50) (3/16) (0/16)
Early HCC
Sensitivity
Specificity
PPV
NPV
Accuracy
66.7% 69.0% 22.7% 80.0% 20.8% 88.2% 23.5% 80.0% 50%
66.7% 69.0% 22.7% 80.0% 20.8% 88.2% 23.5% 80.0% 50.0%
100.0% 98.0% 90.8% 84.5% 95.9% 78.0% 100.0% 81.2% 100.0%
100.0% 90.9% 35.7% 65.1% 76.9% 57.7% 100.0% 88.9% 100.0%
69.2% 91.4% 84.0% 92.1% 64.8% 95.1% 79.4% 68.4% 51.6%
81.0% 91.3% 78.3% 83.3% 66.1% 80.6% 80.6% 80.4% 67.4%
(8/12) (20/29) (5/22) (28/35) (10/48) (15/17) (4/17) (24/30) (15/30)
HGDN, high-grade dysplastic nodule; HCC, hepatocellular carcinoma; PPV, positive predictive value; NPV, negative predictive value; DCP, des-gamma-carboxy prothrombin; HSP70, heat shock protein 70; GPC3, glypican-3; GS, glutamine synthetase; CHC, clathrin heavy chain.
Table 2
Staging: tumour staging is the most important prognostic factor for all malignant tumours. It assists oncologists in predicting clinical outcome, choice of appropriate treatment, and estimation of therapeutic effects. The American Joint Committee on Cancer (AJCC) Tumour-Node-Metastasis (TNM) system is the major and most commonly used staging system for HCC. As pathologists, we apply the TNM classification to resected tumours; solitary HCC of any size is stage pT1, while an HCC less than 5 cm diameter but multifocal and/or with vascular invasion is pT2. Thus histologically early HCC may be stage pT1 or 2. However, treatment selection for HCC patients considers ablative as well as surgical management and requires a staging system based not only on tumour extent, but also underlying liver disease, cirrhotic status, and liver functional reserve. Several clinical staging systems, such as the Okuda classification, the Cancer of the Liver Italian Program (CLIP) system, the Barcelona-Clinic Liver Cancer (BCLC) system, and the Chinese University Prognostic Index (CUPI), have been developed to attempt better stratification of HCCs.46 There is no worldwide consensus on the use of such staging systems, nor consistent results in comparing the performance of different systems in a variety of cohorts. Among these staging systems, the BCLC system is endorsed by AASLD and EASL, externally validated by several groups, and widely applied in major clinical trials.4,5,46 It incorporates tumour characteristics (tumour size, number of nodules, and presence of portal vein thrombosis), liver function (ChildePugh score, portal hypertension, bilirubin level), physical status and cancer-related symptoms. It stratifies HCC into five clinicopathological stages, namely 0 (very early), A (early), B (intermediate), C (advanced) and D (terminal) with corresponding treatment recommendations. Stages A (early) and 0 (very early) are not equivalent to pathologically defined early HCC. Early stage HCC includes asymptomatic patients with preserved liver function (ChildePugh A and B) and a single HCC smaller than 5 cm or up to three nodules smaller than 3 cm. Very early stage HCC refers to asymptomatic patients with well-preserved liver function (ChildePugh A) and a solitary HCC less than 2 cm. The histopathological concepts of ‘early HCC’ that form the basis of this article are not currently
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incorporated into clinicopathological staging systems for HCC. Therefore careful communication is needed to distinguish between histological and clinical ‘early’ HCC.
Conclusion Early HCC exhibits characteristic pathological, radiological and molecular features. Pathologists play an important role in the accurate diagnosis of early HCC by detailed morphological examination (see Table 1) and careful selection of tumour markers (Table 2). The limitations of current morphological criteria and existing tumour markers are accepted. Timely recognition of early HCC and translation to clinical practice are challenging to clinicians, radiologists and pathologists, and require more systematic clinical studies and trials in the near future. A
REFERENCES 1 Ferlay J, Shin HR, Bray F, et al. GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer. Available from: http:// globocan.iarc.fr; 2010. 2 Omata M, Lesmana LA, Tateishi R, et al. Asian Pacific Association for the Study of the Liver consensus recommendations on hepatocellular carcinoma. Hepatol Int 2010; 4: 439e74. 3 Venook AP, Papandreou C, Furuse J, de Guevara LL. The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective. Oncologist 2010; 15(suppl 4): 5e13. 4 Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: 421e30. 5 Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005; 42: 1208e36. 6 Anthony PP, Vogel CL, Barker LF. Liver cell dysplasia: a premalignant condition. J Clin Pathol 1973; 26: 217e23. 7 Lee RG, Tsamandas AC, Demetris AJ. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched case-
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28 Nascimento C, Caroli-Bottino A, Paschoal J, Pannain VL. Vascular immunohistochemical markers: contributions to hepatocellular nodule diagnosis in explanted livers. Transplant Proc 2009; 41: 4211e3. 29 Park YN, Kim YB, Yang KM, Park C. Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch Pathol Lab Med 2000; 124: 1061e5. 30 Chuma M, Sakamoto M, Yamazaki K, et al. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology 2003; 37: 198e207. 31 Lim SO, Park SG, Yoo JH, et al. Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in hepatitis B virusrelated hepatocellular carcinomas and dysplastic nodules. World J Gastroenterol 2005; 11: 2072e9. 32 Di Tommaso L, Destro A, Seok JY, et al. The application of markers (HSP70 GPC3 and GS) in liver biopsies is useful for detection of hepatocellular carcinoma. J Hepatol 2009; 50: 746e54. 33 Cai MY, Tong ZT, Zheng F, et al. EZH2 protein: a promising immunomarker for the detection of hepatocellular carcinomas in liver needle biopsies. Gut 2011; 60: 967e76. 34 Shibata R, Mori T, Du W, et al. Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis. Clin Cancer Res 2006; 12: 5363e8. 35 Llovet JM, Chen Y, Wurmbach E, et al. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology 2006; 131: 1758e67. 36 Cheng W, Tseng CJ, Lin TT, et al. Glypican-3-mediated oncogenesis involves the insulin-like growth factor-signaling pathway. Carcinogenesis 2008; 29: 1319e26. 37 Okabe H, Satoh S, Kato T, et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression. Cancer Res 2001; 61: 2129e37. 38 Christa L, Simon MT, Flinois JP, et al. Overexpression of glutamine synthetase in human primary liver cancer. Gastroenterology 1994; 106: 1312e20. 39 Cadoret A, Ovejero C, Terris B, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002; 21: 8293e301. 40 Osada T, Sakamoto M, Nagawa H, et al. Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis. Cancer 1999; 85: 819e31. 41 Long J, Lang ZW, Wang HG, et al. Glutamine synthetase as an early marker for hepatocellular carcinoma based on proteomic analysis of resected small hepatocellular carcinomas. Hepatobiliary Pancreat Dis Int 2010; 9: 296e305. 42 Di Tommaso L, Franchi G, Park YN, et al. Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis. Hepatology 2007; 45: 725e34. 43 Di Tommaso L, Destro A, Fabbris V, et al. Diagnostic accuracy of clathrin heavy chain staining in a marker panel for the diagnosis of small hepatocellular carcinoma. Hepatology 2011; 53: 1549e57. 44 Yu NC, Chaudhari V, Raman SS, et al. CT and MRI improve detection of hepatocellular carcinoma, compared with ultrasound
control study, pathological analysis, and pathogenetic hypothesis. Hepatology 1997; 26: 1415e22. Marchio A, Terris B, Meddeb M, et al. Chromosomal abnormalities in liver cell dysplasia detected by comparative genomic hybridisation. Mol Pathol 2001; 54: 270e4. Park YN. Update on precursor and early lesions of hepatocellular carcinomas. Arch Pathol Lab Med 2011; 135: 704e15. Watanabe S, Okita K, Harada T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983; 51: 2197e205. Plentz RR, Park YN, Lechel A, et al. Telomere shortening and inactivation of cell cycle checkpoints characterize human hepatocarcinogenesis. Hepatology 2007; 45: 968e76. Deugnier YM, Charalambous P, Le Quilleuc D, et al. Preneoplastic significance of hepatic iron-free foci in genetic hemochromatosis: a study of 185 patients. Hepatology 1993; 18: 1363e9. Asare GA, Paterson AC, Kew MC, Khan S, Mossanda KS. Iron-free neoplastic nodules and hepatocellular carcinoma without cirrhosis in Wistar rats fed a diet high in iron. J Pathol 2006; 208: 82e90. Libbrecht L, Desmet V, Roskams T. Preneoplastic lesions in human hepatocarcinogenesis. Liver Int 2005; 25: 16e27. Terminology of nodular hepatocellular lesions. International Working Party. Hepatology 1995; 22: 983e93. Pathologic diagnosis of early hepatocellular carcinoma: a report of the international consensus group for hepatocellular neoplasia. Hepatology 2009; 49: 658e64. Takayama T, Makuuchi M, Hirohashi S, et al. Early hepatocellular carcinoma as an entity with a high rate of surgical cure. Hepatology 1998; 28: 1241e6. Park YN, Kojiro M, Di Tommaso L, et al. Ductular reaction is helpful in defining early stromal invasion, small hepatocellular carcinomas, and dysplastic nodules. Cancer 2007; 109: 915e23. Tateishi R, Yoshida H, Matsuyama Y, et al. Diagnostic accuracy of tumor markers for hepatocellular carcinoma: a systematic review. Hepatol Int 2008; 2: 17e30. Fujioka M, Nakashima Y, Nakashima O, Kojiro M. Immunohistologic study on the expressions of alpha-fetoprotein and protein induced by vitamin K absence or antagonist II in surgically resected small hepatocellular carcinoma. Hepatology 2001; 34: 1128e34. Malaguarnera G, Giordano M, Paladina I, et al. Serum markers of hepatocellular carcinoma. Dig Dis Sci 2010; 55: 2744e55. Kumada T, Nakano S, Takeda I, et al. Clinical utility of Lens culinaris agglutinin-reactive alpha-fetoprotein in small hepatocellular carcinoma: special reference to imaging diagnosis. J Hepatol 1999; 30: 125e30. Tamano M, Sugaya H, Oguma M, et al. Immunolocalisation of PIVKA-II in paraffin-embedded specimens of hepatocellular carcinoma. Liver 1999; 19: 406e10. Miskad UA, Yano Y, Nakaji M, et al. Histological study of PIVKA-II expression in hepatocellular carcinoma and adenomatous hyperplasia. Pathol Int 2001; 51: 916e22. Park YN, Yang CP, Fernandez GJ, et al. Neoangiogenesis and sinusoidal “capillarization” in dysplastic nodules of the liver. Am J Surg Pathol 1998; 22: 656e62. de Boer WB, Segal A, Frost FA, Sterrett GF. Can CD34 discriminate between benign and malignant hepatocytic lesions in fine-needle aspirates and thin core biopsies? Cancer 2000; 90: 273e8. Coston WM, Loera S, Lau SK, et al. Distinction of hepatocellular carcinoma from benign hepatic mimickers using Glypican-3 and CD34 immunohistochemistry. Am J Surg Pathol 2008; 32: 433e44.
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alone, in patients with cirrhosis. Clin Gastroenterol Hepatol 2011; 9: 161e7. 45 Forner A, Vilana R, Ayuso C, et al. Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: Prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma. Hepatology 2008; 47: 97e104. 46 Marrero JA, Kudo M, Bronowicki JP. The challenge of prognosis and staging for hepatocellular carcinoma. Oncologist 2010; 15(suppl 4): 23e33.
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Large cell change is often a degenerative senescent change, which may be best regarded as a predictive marker associated with HCC, while small cell change and iron-free foci are considered as genuine premalignant lesions of HCC Morphological distinction between low-grade dysplastic nodules and large regenerative nodules is impossible by current consensus. It has been argued that the latter term should be dropped
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Atypical architectural and/or cytological features differentiate high-grade dysplastic nodules from low-grade dysplastic nodules There is a marked overlap in pathological and radiological features between early HCC and high-grade dysplastic nodules. The inter-observer agreement is still suboptimal among the experts in the current consensus Stromal invasion, which is defined as infiltration of tumour cells into intratumoural portal tracts or fibrous septa, is the key distinctive feature of early HCC from high-grade dysplastic nodule. Absence of significant ductular reaction (with the aid of immunostaining of cytokeratins 7/19) favours genuine stromal invasion rather than pseudoinvasion Complete sinusoidal staining of CD34 is useful in differentiating early HCC from high-grade dysplastic nodules with >90% positive/negative predictive values and accuracy The applications of various conventional and emerging potential diagnostic markers in assisting pathological diagnosis of early HCC need further investigation None of currently available staging systems uses tumour histology to accurately categorize early HCC
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Liver cell dysplasia and early hepatocellular carcinoma
increased in “low-risk” regions. The incidence in Hong Kong and Japan for example dropped by 35.1% and 42.7% respectively in the last two decades. This reduction is largely a result of Hepatitis virus B vaccination programs initiated in many East Asian and Asia-Pacific countries during the late 1980s.2 In contrast, the incidence has tripled in the United States between 1975 and 2005; similar rising trends are observed in the United Kingdom, Canada and Australia. Such growth is attributable to an epidemic of chronic hepatitis virus C infection, an increasingly young onset of alcohol consumption, and rising rates of obesity and diabetes mellitus with a consequent increase in prevalence of nonalcoholic steatohepatitis.3 Primary prevention through the control of risk factors contributing to the development of HCC is crucial for dealing with this challenging malignancy of global importance. Secondary prevention (i.e. interventions leading to the discovery and control of cancerous or precancerous processes while localized) is equally important to allow early diagnosis and prompt treatment of HCC to improve clinical outcome. Much of the high fatality rate of HCC (0.93 ratio of mortality to incidence) is due to the late detection of advanced HCC. Surveillance programs for at-risk populations have now been widely established.2,4,5 Advances in radiological techniques in such screening allow early detection of small hepatic nodules less than 2 cm in patients with chronic liver diseases. These small hepatic nodules represent a broad-spectrum of entities from benign, premalignant, to early malignant lesions. The accurate characterization of premalignant and early lesions of HCC poses a significant diagnostic challenge. The terminology for these lesions is based on a 1995 International Working Party (IWP) and is described in full below. Definition of the entities is important. The histological diagnosis of a macroscopically distinct small nodular lesion seen on imaging and on the cut surface of the subsequent resection specimens will be one of the following: large regenerative nodule, low-grade dysplastic nodule, highgrade dysplastic nodule, early HCC, progressed HCC (Table 1). There are other terms which refer to microscopic abnormalities that may be diagnostic features of the above; these include cytological changes in hepatocytes (large cell change, LCC; small cell change), and expansile foci of such cytological change (dysplastic foci). A histologically early HCC may have an indistinct margin macroscopically, and the term ‘vaguely nodular’ has been used for such lesions.
Anthony W H Chan Alastair D Burt
Abstract Hepatocellular carcinoma (HCC) is a major world health problem. Screening and diagnosis of early lesions of HCC allows prompt treatment to improve clinical outcomes. Early lesions of HCC include premalignant lesions and small HCCs. The former consists of hepatocellular cytological changes (large cell change, small cell change and iron-free foci), dysplastic foci and dysplastic nodules (low-grade and high-grade); while the latter is subdivided into early HCC and progressed HCC. There is significant overlap in pathological and radiological features between high-grade dysplastic nodules and early HCC making a precise diagnosis demanding even among experts. Various conventional and emerging potential diagnostic markers have been evaluated in assisting pathological diagnosis of early HCC. Translation of the concept of early HCC to daily clinical practice in term of diagnosis and tumour staging is challenging to clinicians, radiologists and pathologists.
Keywords dysplasia; hepatocellular carcinoma; liver; liver cancer
Introduction Hepatocellular carcinoma (HCC) is a major world health problem with a high incidence and fatality rate. Based on the latest data from GLOBOCAN by the International Agency for Research on Cancer, HCC is the fifth commonest cancer in men (523,000 cases, 7.9% of the total) and the seventh in women (226,000 cases, 6.5% of the total) worldwide.1 The majority (85%) of the cases occur in developing countries in Eastern and Southeast Asia, Middle and Western Africa. The annual age-standardized incidence rate in Eastern, South-Eastern Asia and Pacific regions is 21.8/100,000 with the highest rate in Mongolia (94.4/ 100,000), while it is much lower in North America (4.4/100,000) and the European Union (4.7/100,000). HCC leads to 477,000 and 217,000 annual deaths in men and women respectively around the World and is the third commonest cause of cancer death; the geographical distribution of the mortality matches that observed for incidence. The epidemiology of HCC has changed over recent decades. The incidence of HCC has decreased in “high-risk” regions but
Premalignant lesions in human hepatocarcinogenesis The development of HCC (hepatocarcinogenesis) is a multistep process characterized by progressive accumulation and interplay of genetic and epigenetic changes leading to uncontrolled clonal proliferation, local invasion and distant metastasis. During the process, a number of phenotypic changes arise, often designated as premalignant lesions.
Anthony W H Chan FRCPA is at the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. Conflicts of interest: none.
Microscopic changes, including dysplastic foci Large cell change, formerly known as large cell dysplasia, was first described by Anthony et al. and initially referred to “liver cell dysplasia” in 1973.6 It is characterized by a group of hepatocytes with cellular and nuclear enlargement, nuclear pleomorphism and multinucleation, prominent nucleoli, and
Alastair D Burt FRCPath is Professor at the Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. Conflicts of interest: none.
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Summary of pathological and radiological features of small nodular lesions in the cirrhotic liver15,16 LRN
Pathological appearance (gross) Pathological appearance (microscopic) Nodule-in-nodule Cell crowding, compared with background cellularity Clone-like population Thickened trabeculae Pseudoglands Deficiency of reticulin fibres Intranodular portal tract Unpaired arteriole Stromal invasion Vascular invasion Nuclear atypia Radiological appearance Arterial supply Isovascular/ hypovascular Portal venous supply
þ
LGDN
HGDN
Early HCC (WD-HCC)
Progressed HCC (MD-HCC)
Usually distinctly nodular
Usually vaguely nodular
Vaguely nodular
Distinctly nodular
(<1.5)
þ (1.5e2)
þ (>2)
þ (>2)
(2 cells thick)
þ (2e3 cells thick) þ
þ (3 cells thick) þ
þ (3 cells thick) þ
þ
þ þ
þ þ þ
þ þ
Isovascular/ hypovascular
Isovascular/ hypovascular
Hypervascular
þ
þ
Isovascular/ hypovascular/rarely hypervascular þ
LRN, large regenerative nodule; LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule; HCC, hepatocellular carcinoma; WD, well-differentiated; MD, moderately differentiated; , absent; , may be present but not necessarily detected in core biopsy; þ, usually present and detected in core biopsy.
Table 1
LCC compared to adjacent HCC.7,8 It is currently believed that LCC usually represents the pathophysiological nuclear polypoidy which is a degenerative senescent change as a consequence of chronic liver injury. In this respect, LCC is best regarded as a predictive marker associated with HCC rather than a genuine premalignant lesion. However, recent data suggests that LCC may, in some situations, be a genuine premalignant lesion.9 Small cell change was initially described by Watanabe et al. as “liver cell dysplasia of small-cell type” in 1983.10 It is characterized by a collection of hepatocytes with a decreased cell volume, minimal nuclear pleomorphism, an increased N:C ratio, and increased nuclear density (Figure 2). It is generally believed to be an early premalignant lesion because of a higher prevalence in cirrhotic livers with HCC than in those without, a high proliferative activity, a morphological continuum between small cell change and HCC, a similar immunophenotype to hepatic progenitor cells, and similar chromosomal alterations in small cell change and adjacent HCC. Plentz et al. demonstrated that markedly reduced expression of p16 and p21 coincided with further telomere shortening and an accumulation of DNA damage in small cell change and HCC compared to LCC and cirrhotic nodules suggesting that small cell change represents a more “advanced” lesion than LCC.11 Iron-free foci in genetic haemochromatosis were shown to be more frequent in livers with HCC (50.0%) than those without (8.3%), and demonstrated a high proliferative index and co-existing LCC/small cell change (71.4%).12 HCCs in genetic
preserved nucleocytoplasmic (N:C) ratio (Figure 1). It was initially considered a premalignant lesion because of a frequent association with HCC and the presence of an abnormal and increased DNA content. However, this concept was challenged by observations of a normal N:C ratio, low proliferative index, high apoptotic activity, lack of a direct transition from LCC to carcinoma, and the absence of relevant genetic abnormalities in
Figure 1 Large cell change featured by cellular and nuclear enlargement, nuclear pleomorphism and multinucleation, prominent nucleoli, and preserved N:C ratio (H&E, original magnification 400).
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Figure 2 Small cell change featured by decreased cell volume, minimal nuclear pleomorphism, an increased N:C ratio, and increased nuclear density (H&E, original magnification 200).
Figure 3 Oncocytic change featured by abundant eosinophilic granular cytoplasm (H&E, original magnification 200).
increased cell density in HGDN can be sometimes up to twice that of the surrounding liver. Both also often exhibit features suggestive of a clonal origin, such as iron or copper accumulation, or steatosis in the context of a background liver without significant fatty change. LGDN is usually distinct rather than vaguely nodular due to the presence of a condensed fibrous scar or pseudocapsule at its periphery. Architectural atypia is absent, while cytological atypia is minimal with occasional LCC and absent mitoses. It often contains intranodular portal tracts, with only very rare aberrant unpaired or non-triad arterioles. Its blood supply is entirely derived from portal veins, and hence it is either hypovascular or isovascular in the arterial phase of contrast-enhanced imaging. It is impossible to confidently distinguish between LGDN and LRN by morphology alone in the current consensus and it has been argued that the latter term should be dropped. HGDN is usually vaguely nodular and contains significant architectural and/or cytological atypia. Atypical architectural features include nodule-in-nodule pattern (Figure 4), pseudoglandular or acinar formation (Figure 5), and thickened trabeculae up to three cells thick. Cytological changes comprise focal or diffuse small cell change, iron-free foci, focal steatosis with Mallory-Denk bodies (Figure 6) or p62 inclusions (Figure 7), and clear cell change. Among all these atypical changes a nodule-in-nodule pattern is the most worrying feature because HCC not uncommonly emerges from such “sub-nodules”. HGDN contains fewer intranodular portal tracts and more aberrant unpaired arterioles than LGDN. Its main blood supply is still derived from the portal vein and hence it is either hypovascular or isovascular on imaging.
haemochromatosis are typically iron-free, and the sequential development from iron-free nodules to iron-free HCC in a recent rat model further support that iron-free foci are premalignant lesions.13 However, the underlying mechanisms of iron resistance in these foci and subsequent progression into HCC remain unknown. A dysplastic focus was defined by the IWP as a microscopic abnormality less than 1 mm in diameter, consisting of an expansile group of hepatocytes with cytological features suggestive of premalignant change, particularly small cell change and iron-free foci. Apart from these well-documented atypias, there are other hepatocellular cytological changes, collectively termed “foci of altered hepatocytes (FAH)”, described in several animal models in early hepatocarcinogenesis caused by chemicals, radiation, and chronic infection with hepadnaviruses.14 Although some FAH, including glycogen-storing clear cell foci, clear cellpredominated mixed cell foci and oncocytic foci (Figure 3), have been also observed in chronic liver disease with or without HCC in humans, there is insufficient evidence to establish the definite role of FAH in human hepatocarcinogenesis. Large regenerative nodules, and dysplastic nodules In the 1995 paper published by the International Working Party (IWP) of the World Congresses of Gastroenterology the following consensus nomenclature and morphological criteria for hepatocellular nodular lesions was proposed15: A large regenerative nodule (LRN) (or macroregenerative nodule) was seen as a nodule with a minimum diameter of 5 mm, and one which is more distinctive and larger than surrounding cirrhotic regenerative nodules. It is composed of hepatocytes that are cytologically bland without any architectural or cytological atypia. It is commonly situated adjacent to a large portal tract. A dysplastic nodule (DN) was defined as a nodule macroscopically distinct from surrounding cirrhotic regenerative nodules due to its size (varied from few millimetres to few centimetres, mostly less than 15 mm), colour, texture, and degree of bulging. It is further classified into low-grade (LGDN) and highgrade (HGDN) according to the degree of architectural and/or cytological atypia. Both LGDN and HGDN are hypercellular;
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Small HCC, early HCC and progressed HCC Small HCC was defined by the IWP as an HCC less than 2 cm, and subdivided into two groups, early HCC and progressed HCC.16,17 Early HCC is also known as “small HCC of vaguely nodular type” and “small HCC with indistinct margin”. Pathologically, this is a well-differentiated HCC with vague nodulation, indistinct borders and various combinations of (1) increased cell density more than twice that of the surrounding liver; (2) small cell change; (3) pseudoglandular pattern; (4) irregular trabecular
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Figure 4 Nodule-in-nodule pattern in a high-grade dysplastic nodule (Reticulin, original magnification 25).
Figure 6 Mallory-Denk bodies. Ropey deeply eosinophilic cytoplasmic inclusions (H&E, original magnification 200).
Progressed HCC is also termed “small HCC of distinct nodular type” and “small nodular HCC with distinct margins”. Pathologically, it is a moderately differentiated HCC with a distinctly nodular appearance often with a fibrous pseudocapsule. It resembles classical HCC with typical trabecular and pseudoglandular architectures, numerous aberrant unpaired arterioles, and absence of intratumoural portal tracts. Vascular invasion and intrahepatic metastasis are found in 29% and 21% of cases respectively.17 Radiologically, progressed HCC is hypervascular as it derives blood exclusively from the arterial supply as classical HCC does. In addition to differences in the pathological and radiological appearances between early HCC and progressed HCC, the latter shows a less favourable outcome than early HCC with 5year overall survivals of 54% and 93% respectively.17
pattern with occasional more than three cells thick; (5) diffuse fatty change; (6) intratumoural portal tracts; and (7) unpaired arterioles. Vascular invasion and intrahepatic metastasis are absent or exceptionally rare. Radiologically, early HCC is hypovascular, isovascular, or very rarely hypervascular. Marked overlapping in pathological and radiological features between early HCC and HGDN makes a precise diagnosis demanding. The key pathological feature for distinguishing early HCC from HGDN is the presence of stromal invasion, which is defined as infiltration of tumour cells into intratumoural portal tracts or fibrous septa (Figure 8). When stromal invasion is difficult to establish unequivocally on routine histology, the absence of significant ductular reaction (with the aid of immunostaining of cytokeratins 7/19) favours genuine stromal invasion rather than pseudoinvasion.18 Another histological investigation that may be helpful is the reticulin stain; convincing deficiency of a reticulin framework in the liver cell plates is a useful diagnostic feature of HCC, but this is not necessarily deficient, and so a normalappearing reticulin does not exclude early HCC.
Updated international consensus and discrepancy between east and west In order to achieve a refined and updated international consensus of hepatic nodules, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in 2002
Figure 5 Pseudoglandular pattern in a high-grade dysplastic nodule (H&E, original magnification 100).
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Figure 7 p62 inclusions. Large oval-shaped deeply eosinophilic cytoplasmic inclusions (H&E, original magnification 200).
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for AFP. Although AFP is a highly specific (95e100%) tissue marker, it is not an optimal diagnostic marker because of low sensitivity (25e30%) and a heterogeneous patchy staining pattern. Its application in early HCC detection is further hindered as Fujioka et al. found that none of 13 cases of early HCC were immunoreactive for AFP.20 AFP can be fractionated by affinity electrophoresis into three glycoforms: L1, L2, and L3 based on the reactivity with the lectin Lens culinaris agglutinin. AFP-L1 is the major glycoform in serum of patients with chronic hepatitis and cirrhosis, while AFPL2 and AFP-L3 are glycoforms more specifically produced by yolk sac tumours and HCC respectively. The percentage of AFPL3 over total AFP level (AFP-L3%) is not only a highly specific (>93%) serum marker for HCC, but also an adverse prognostic marker associated with vascular invasion and intrahepatic metastasis.19,21 Although the detection rate of small HCC by AFPL3% is low (22.2e23.0%), there is a potential value for differentiation between early HCC and progressed HCC.19,22 Kumada et al. found that none of 15 cases (0%) with early HCC and 14 of 45 cases (31.1%) with progressed HCC showed diagnostic levels of AFP-L3%.22 An antibody against AFP-L3 for immunohistochemistry however is not yet commercially available. Des-gamma-carboxy prothrombin (DCP), also known as protein induced by vitamin K absence or antagonist-II (PIVKAII), is an abnormal prothrombin produced by HCC tumour cells. It has been used as a specific serum marker, as well as a poor prognostic marker because of its association with early portal vein invasion, intrahepatic metastasis and capsular infiltration. Compared to serum AFP in the detection of small HCC, it offers comparable sensitivity (44e54%) and higher specificity (95e 99%).21 Few studies have investigated tissue DCP expression by immunohistochemistry; these demonstrated unsatisfactory sensitivities of 63.2% (50.0e81.8%), 48.1% (29.6e75%) and 47.8% (39.3e61.0%) in identifying all, well-differentiated and small HCCs respectively.20,23,24 Miskad et al. found that eight of 12 early HCCs (66.7%) and none of nine DNs (0%) were immunoreactive for DCP, which could be used as a potential diagnostic aid.24 CD34 is an intercellular adhesion glycoprotein and one of the commonly used vascular endothelial markers. Sinusoidal endothelium in normal liver is devoid of CD34 immunoreactivity. However, many non-neoplastic and neoplastic liver diseases lead to distortion of the normal hepatic microcirculation and the formation of aberrant new vessels (neo-vascularization), resulting in sinusoidal capillarization. Two distinct patterns of capillarization can be demonstrated by immunostaining of CD34 e complete and incomplete patterns. The former is characterized by a diffuse sinusoidal expression of CD34 within the entire lesion, while an incomplete pattern is featured by focal and preferentially peripheral sinusoidal expression of CD34 in the lesion. The complete CD34 staining pattern is sensitive in detecting 93.2% (82.5e100%) and 89.8% (82.7e100%) of all and well-differentiated HCCs respectively with a high specificity of 96.2% (82.5e100%).25e28 It is also observed in 3.7% of FNH and 20.7% of HCA but this does not create any major diagnostic issues when tackling a nodular lesion in a cirrhotic liver together with radiological correlation. Cirrhotic nodules, LRN and LGDN do not exhibit the complete pattern. With respect early HCC and its major mimicker HGDN, complete CD34 staining is observed in
Figure 8 Stromal invasion in an early HCC. Infiltration of tumour cells into intratumoural portal tracts without accompanied ductular reaction (H&E, original magnification 100).
with several subsequent meetings until 2007.16 The latest ICGHN comprised of 34 hepatopathologists and two clinicians from 13 countries, including most members of the original IWP who are still active and all the participants from the first ICGHN meeting. A total of 26 surgically resected nodules from 23 patients with chronic hepatitis B or C were selected from Japanese and Korean medical centres; all the nodules were less than 2 cm. For each case, all pathologists in the study group independently reviewed brief clinical data, a gross picture, and an H&E slide containing the entire width of the lesion, and classified the lesion according to the IWP criteria. There was little disparity in distinguishing early HCC/HGDN from LGDN/LRN or progressed HCC. However, the differentiation between early HCC and HGDN showed a significant disagreement between Eastern and Western pathologists. The former had much higher tendency to diagnose early HCC. At the first conference, the diagnosis of early HCC and HGDN showed wide inter-observer variation with very low Kappa value of 0.30. By the addition of clearly defined stromal invasion to the diagnostic criteria for early HCC the discrepancy decreased and the Kappa value rose to 0.49 at the subsequent conference. However, the inter-observer agreement is still suboptimal and further fine-tuning of the diagnostic criteria and exploring suitable diagnostic markers are necessary.
Early HCC and conventional diagnostic markers There are several well-established serum and tissue markers for HCC. Alpha-fetoprotein (AFP) is a major plasma glycoprotein produced by the yolk sac and liver during normal foetal development. It has been used as a serum marker for HCC since the 1970s. However, serum AFP as a diagnostic/screening test for small HCC is of limited value. Serum AFP has a sensitivity of only 49e51% and a specificity of 83e86% in the detection of small HCC.19 Tissue AFP expression demonstrated by immunohistochemistry in a hepatic lesion is diagnostic of malignant hepatocellular neoplasm (HCC or hepatoblastoma) excluding rare metastatic yolk sac tumour or metastatic hepatoid carcinoma. Other HCC mimickers including LRN, DN, focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are all negative
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69.0% and 2.0% respectively, and therefore this could serve as a useful diagnostic tool.27,29
Glutamine synthetase (GS) catalyzes the synthesis of glutamine, the major energy source of tumour cells. It plays an important role in ammonia detoxification, nitrogen homeostasis, and acidebase balance in the mammalian liver. Christa et al. showed up-regulation of GS mRNA, protein, and enzyme activity in HCC, and subsequently Cadoret et al. observed the activation of Wnt/beta-catenin pathway leading to this overexpression.38,39 Osada et al. demonstrated a progressive increase in GS immunoreactivity from DN, early HCC to advanced HCC, suggesting an involvement in early hepatocarcinogenesis.40 A recent proteomic analysis of HCC less than 3 cm consolidated the role of GS in early hepatocarcinogenesis.41 Immunoreactivity of GS is observed in 44.8% and 20.8% of all and early HCCs respectively, compared with 4.1%, 0% and 0% of HGDN, LGDN and cirrhosis respectively. Similarly to HSP70, its application in differentiation between early HCC and HGDN is limited by low sensitivity and accuracy.32,40 A panel of three markers, GPC3, GS and HSP-70 is currently emerging as a helpful strategy for diagnosing HCC. Positivity for any two of the three markers was found to indicate malignancy with 72% sensitivity and 100% specificity.42 When the same panel was used to retrospectively distinguish between HCC and HGDN on liver biopsies, the sensitivity decreased to 49% while the specificity remained 100%.32 A recent study suggested that the addition of clathrin heavy chain (CHC) to the existing panel of three markers produced an improvement in the sensitivity and diagnostic accuracy when two markers were positive whilst still retaining the 100% specificity.43
Early HCC and emerging potential diagnostic markers With a better understanding of genetic and epigenetic changes in early HCC through different high throughput genome-wide and other novel emerging molecular methodologies, we may have a better insight of early hepatocarcinogenesis and the identification of potential diagnostic markers for detection of early HCC. Heat shock protein 70 (HSP70) is a family of proteins implicated in carcinogenesis, regulation of cell-cycle progression and apoptosis. Many HCCs arise from liver diseases with chronic necroinflammation and fibrosis, which induce the synthesis of such heat shock proteins. Chuma et al. employed oligonucleotide arrays of 12,600 genes and demonstrated that HSP70 is the most abundantly up-regulated gene in early HCC. This study also found a progressive increase in messenger RNA (mRNA) levels and tissue expression of HSP70 from LGDN, HGDN, early HCC to progressed HCC by using quantitative realtime reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry respectively.30 Subsequent studies by immunohistochemistry from other groups confirmed this finding, showing overexpression of HSP70 in 58.7%, 46.7% and 22.7% of all, well-differentiated and early HCCs respectively, in contrast to 9.2%, 0% and 0% of HGDN, LGDN and cirrhosis respectively.31e33 However, HSP70 immunohistochemistry, its application in differentiation between early HCC and HGDN is restricted by its low sensitivity and positive predictive value.31,32 Cyclase-associated protein 2 (CAP2) is a protein involved in the interaction between adenylyl cyclase and actin but its exact function in human remains unknown. In the same genome-wide study of early HCC of Chuma et al., CAP2 was another upregulated gene.30 The follow-up study by the same group demonstrated through qRT-PCR and immunohistochemistry stepwise increments in CAP2 mRNA and protein levels from DN, early HCC to progressed HCC.34 Although this is a promising diagnostic marker for early HCC, there are no other studies yet available to validate the findings. Glypican-3 (GPC3) is a member of glycosyl-phosphatidylinositol anchored heparin sulphate proteoglycans. It is normally expressed in foetal but not adult liver. It promotes the growth of HCC tumour cells by activating Wnt/beta-catenin and insulin-like growth factor signalling pathways.35,36 Okabe et al. identified GPC3 being one of 86 up-regulated genes in HCC by using complementary DNA microarrays consisting of 23,040 genes.37 Llovet et al. further demonstrated GPC3 to be one of five upregulated genes in early HCC compared to DN by analyzing transcriptional profiles of 55 candidate genes through qRT-PCR, and showed tissue expression of GPC3 by immunohistochemistry in HCC but not DN.35 Several immunohistochemical studies illustrated that GPC3 is sensitive in detecting 78.6% (68.1e88.8%) and 68.8% (50.0e94.4%) of all and welldifferentiated HCCs respectively with the overall specificity of 92.9% (87.0e98.2%).27,32,33 It is of potential diagnostic use because of a significantly different expression in early HCC (80.0%) compared with HGDN (15.5%).27,32
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Clinical implications of early HCC Diagnosis: the apparently favourable prognosis of early HCC emphasizes its importance in incorporation into clinical practice. Ultrasonography (USG) is the radiological test for surveillance for HCC in cirrhotic patients recommended by the European Association for the Study of the Liver (EASL), the American Association for the Study of Liver Diseases (AASLD) and the AsianPacific Association for the Study of the Liver (APASL).2,4,5 However, the USG detection rate drops significantly from 65 e80% of conventional HCC to 21% of small HCC.5,44 To establish a definitive radiological diagnosis of small HCC, other modalities including contrast-enhanced USG and magnetic resonance imaging remain suboptimal with unsatisfactory sensitivity of 61.7% and 57.1% respectively.45 The major underlying hindrance is the hypovascularity of early HCC. AASLD recommends liver biopsy for hepatic nodules between 1 and 2 cm without a characteristic radiological vascular pattern of HCC.5 However, a confident histopathological distinction between early HCC and HGDN is often impossible because intranodular portal tracts or fibrotic septa (essential for the determination of stromal invasion) may not be present in needle biopsy specimens. Distinguishing LRN/LGDN and progressed HCC arising in pre-existing DN from HGDN/early HCC is sometimes not straightforward because of lesional heterogeneity and sampling error. Nevertheless, liver biopsy remains more sensitive than imaging studies for the detection of early HCC.45 The application of immunohistochemical markers may be helpful, but one should remember that the accuracy of these markers for diagnosis of early HCC in biopsy specimens is not properly validated.
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Diagnostic accuracy of immunohistochemical tumour markers in differentiation between high-grade dysplastic nodule and early hepatocellular carcinoma Tumour marker
HGDN
DCP24 CD34 (complete capillarization)27,29 HSP7031,32 GPC327,32 GS32,40 Any one of HSP70/GPC3/GS32 Any two of HSP70/GPC3/GS32 Any one of HSP/GPC/GS/CHC43 Any two of HSP/GPC/GS/CHC43
0% 2.0% 9.2% 15.5% 4.1% 22.0% 0% 18.8% 0%
(0/9) (2/98) (9/98) (15/97) (3/73) (11/50) (0/50) (3/16) (0/16)
Early HCC
Sensitivity
Specificity
PPV
NPV
Accuracy
66.7% 69.0% 22.7% 80.0% 20.8% 88.2% 23.5% 80.0% 50%
66.7% 69.0% 22.7% 80.0% 20.8% 88.2% 23.5% 80.0% 50.0%
100.0% 98.0% 90.8% 84.5% 95.9% 78.0% 100.0% 81.2% 100.0%
100.0% 90.9% 35.7% 65.1% 76.9% 57.7% 100.0% 88.9% 100.0%
69.2% 91.4% 84.0% 92.1% 64.8% 95.1% 79.4% 68.4% 51.6%
81.0% 91.3% 78.3% 83.3% 66.1% 80.6% 80.6% 80.4% 67.4%
(8/12) (20/29) (5/22) (28/35) (10/48) (15/17) (4/17) (24/30) (15/30)
HGDN, high-grade dysplastic nodule; HCC, hepatocellular carcinoma; PPV, positive predictive value; NPV, negative predictive value; DCP, des-gamma-carboxy prothrombin; HSP70, heat shock protein 70; GPC3, glypican-3; GS, glutamine synthetase; CHC, clathrin heavy chain.
Table 2
Staging: tumour staging is the most important prognostic factor for all malignant tumours. It assists oncologists in predicting clinical outcome, choice of appropriate treatment, and estimation of therapeutic effects. The American Joint Committee on Cancer (AJCC) Tumour-Node-Metastasis (TNM) system is the major and most commonly used staging system for HCC. As pathologists, we apply the TNM classification to resected tumours; solitary HCC of any size is stage pT1, while an HCC less than 5 cm diameter but multifocal and/or with vascular invasion is pT2. Thus histologically early HCC may be stage pT1 or 2. However, treatment selection for HCC patients considers ablative as well as surgical management and requires a staging system based not only on tumour extent, but also underlying liver disease, cirrhotic status, and liver functional reserve. Several clinical staging systems, such as the Okuda classification, the Cancer of the Liver Italian Program (CLIP) system, the Barcelona-Clinic Liver Cancer (BCLC) system, and the Chinese University Prognostic Index (CUPI), have been developed to attempt better stratification of HCCs.46 There is no worldwide consensus on the use of such staging systems, nor consistent results in comparing the performance of different systems in a variety of cohorts. Among these staging systems, the BCLC system is endorsed by AASLD and EASL, externally validated by several groups, and widely applied in major clinical trials.4,5,46 It incorporates tumour characteristics (tumour size, number of nodules, and presence of portal vein thrombosis), liver function (ChildePugh score, portal hypertension, bilirubin level), physical status and cancer-related symptoms. It stratifies HCC into five clinicopathological stages, namely 0 (very early), A (early), B (intermediate), C (advanced) and D (terminal) with corresponding treatment recommendations. Stages A (early) and 0 (very early) are not equivalent to pathologically defined early HCC. Early stage HCC includes asymptomatic patients with preserved liver function (ChildePugh A and B) and a single HCC smaller than 5 cm or up to three nodules smaller than 3 cm. Very early stage HCC refers to asymptomatic patients with well-preserved liver function (ChildePugh A) and a solitary HCC less than 2 cm. The histopathological concepts of ‘early HCC’ that form the basis of this article are not currently
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incorporated into clinicopathological staging systems for HCC. Therefore careful communication is needed to distinguish between histological and clinical ‘early’ HCC.
Conclusion Early HCC exhibits characteristic pathological, radiological and molecular features. Pathologists play an important role in the accurate diagnosis of early HCC by detailed morphological examination (see Table 1) and careful selection of tumour markers (Table 2). The limitations of current morphological criteria and existing tumour markers are accepted. Timely recognition of early HCC and translation to clinical practice are challenging to clinicians, radiologists and pathologists, and require more systematic clinical studies and trials in the near future. A
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alone, in patients with cirrhosis. Clin Gastroenterol Hepatol 2011; 9: 161e7. 45 Forner A, Vilana R, Ayuso C, et al. Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: Prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma. Hepatology 2008; 47: 97e104. 46 Marrero JA, Kudo M, Bronowicki JP. The challenge of prognosis and staging for hepatocellular carcinoma. Oncologist 2010; 15(suppl 4): 23e33.
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Large cell change is often a degenerative senescent change, which may be best regarded as a predictive marker associated with HCC, while small cell change and iron-free foci are considered as genuine premalignant lesions of HCC Morphological distinction between low-grade dysplastic nodules and large regenerative nodules is impossible by current consensus. It has been argued that the latter term should be dropped
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Atypical architectural and/or cytological features differentiate high-grade dysplastic nodules from low-grade dysplastic nodules There is a marked overlap in pathological and radiological features between early HCC and high-grade dysplastic nodules. The inter-observer agreement is still suboptimal among the experts in the current consensus Stromal invasion, which is defined as infiltration of tumour cells into intratumoural portal tracts or fibrous septa, is the key distinctive feature of early HCC from high-grade dysplastic nodule. Absence of significant ductular reaction (with the aid of immunostaining of cytokeratins 7/19) favours genuine stromal invasion rather than pseudoinvasion Complete sinusoidal staining of CD34 is useful in differentiating early HCC from high-grade dysplastic nodules with >90% positive/negative predictive values and accuracy The applications of various conventional and emerging potential diagnostic markers in assisting pathological diagnosis of early HCC need further investigation None of currently available staging systems uses tumour histology to accurately categorize early HCC
Ó 2011 Elsevier Ltd. All rights reserved.