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Impact of large regenerative, low grade and high grade dysplastic nodules in hepatocellular carcinoma development
Journal of Hepatology 39 (2003) 208–214 www.elsevier.com/locate/jhep
Impact of large regenerative, low grade and high grade dysplastic nodules in hepatocellular carcinoma development Mauro Borzio1,2,*, Silvia Fargion3, Franco Borzio1,2, Anna Ludovica Fracanzani3, Anna Maria Croce1,2, Tommaso Stroffolini4, Silvia Oldani1,2, Rodolfo Cotichini5, Massimo Roncalli6 1
Department of Medicine, Gastroenterology Unit, Ospedale Fatebenefratelli, C.so Porta Nuova 23, 20121 Milan, Italy Department of Pathology, Gastroenterology Unit, Ospedale Fatebenefratelli, C.so Porta Nuova 23, 20121 Milan, Italy 3 Department of Internal Medicine, Ospedale Maggiore IRCCS, Universita` di Milano, Milan, Italy 4 Liver Unit, Ospedale San Giacomo, Rome, Italy 5 Laboratory of Epidemiology, Istituto Superiore di Sanita`, Rome, Italy 6 Department of Pathology, Istituto Clinico Humanitas, Rozzano, Universita` di Milano, Milan, Italy
2
See Editorial, pages 269 – 271
Background/Aims: The natural outcome of ultrasound-detected macronodules in cirrhosis is still poorly understood. In this study we assessed the incidence and predictors of malignant transformation in a prospective study of 90 consecutive ultrasound-detected macronodules in cirrhosis. Methods: Macronodules classification was based on recently proposed histological criteria. Extranodular large (LCC) and small cell changes were also evaluated. The follow-up included ultrasound and serum alfa-fetoprotein determination every 3 months. Independent predictors of hepatocellular carcinoma were evaluated by Cox proportional hazards regression analysis. Results: During a mean follow-up of 33 months, 28 (31%) nodules transformed into hepatocellular carcinoma. The incidence of hepatocellular carcinoma per 100 person-years of follow-up was 11.3%, with a malignant transformation rate of 3.5, 15.5, 31 and 48.5% at 1, 2, 3, and 5 years respectively. High-grade dysplastic nodules (HGDN) (hazard risk 5 2.4; CI 95% 5 1.1– 5.0) and LCC (hazard risk 5 3.1; CI 95% 5 1.2 –7.8) were independent predictors of malignant transformation. Eight additional hepatocellular carcinomas developed outside the original lesions raising the overall malignant transformation rate to 40% while 15 macronodules (17%) became undetectable at ultrasound (US). Conclusions: Macronodules characterize a cirrhotic subpopulation with high risk of hepatocellular carcinoma. HGDN and LCC are strong predictors of malignant transformation; subjects with simultaneous presence of both these two conditions are at highest risk of cancer development. The management of cirrhotics with macronodules should be based on morphologic features detected on liver microsamples. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Large regenerative nodule; Low-grade dysplastic nodule; High-grade dysplastic nodule hepatocellular carcinoma; Cirrhosis
1. Introduction Ultrasound detection of small nodular lesions in cirrhosis is becoming a routine daily practice, requiring an accurate Received 2 December 2002; received in revised form 27 March 2003; accepted 9 April 2003 * Corresponding author. Tel.: þ39-2-6363-2596; fax: þ 39-2-6363-2665. E-mail address: [email protected] (M. Borzio).
diagnostic workout. The correct labeling of any hepatic nodule and the number of those truly malignant are essential for a proper clinical management, particularly when surgery and orthotopic liver transplantation (OLT) are available options, given the stringent inclusion criteria recently proposed [1]. The differential diagnosis of small nodular lesions arising in cirrhosis is basically restricted to early hepatocellular carcinoma (HCC) and non-malignant macronodules which
0168-8278/03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-8278(03)00190-9
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include large regenerative, low and high grade dysplastic nodules [2]. To date, the prevailing opinion is that high grade dysplastic nodules are the true HCC precursors while the remaining nodules are though to be only marginally involved in human liver carcinogenesis [3]. Nevertheless, the clinical significance of macronodules detected ‘in vivo’ and their management are still debated and under the focus of extensive research. Indeed, most of the studies addressing their natural outcome were retrospective or mainly restricted to large regenerative or atypical/border-line lesions [4 – 7]. In a recent study of 32 ultrasound (US)-detected hepatocellular nodules, some variables predicting their malignant transformation have emerged but, given the small size of the cohort, results were considered preliminary [8]. In the present study we definitely report on the natural outcome of a large series of macronodules consecutively detected by US, during a program of HCC surveillance in cirrhosis, histologically classified according to the criteria proposed by the International Working Party [2]. The aim of this study was to ascertain the truly malignant potential of the different hepatic macronodules and to establish the predictive significance of additional clinic, histologic and radiologic variables.
2. Materials and methods This study was carried out on 90 consecutive patients (61 male and 29 female) with US-detected macronodules identified at two different Hospitals from 1990 to 1998. Patients were part of a larger series of more than 400 cirrhotics recruited in a screening program for HCC surveillance undergoing US and alfa-fetoprotein (AFP) determination every 6 months. Only patients fulfilling inclusion criteria (see below) were admitted to this study. All focal liver lesions detected during follow-up were histologically examined by an intra and extranodular sampling under US guidance. In case of first negative/unclear biopsy a second biopsy was repeated within 3 months. The histologic diagnosis was also supported by conventional or spiral computed tomography (CT) or MRI or hepatic arteriography plus lipiodol infusion. None of the patients enrolled in this study were treated by interferon after inclusion. The following inclusion criteria were used: (a) histologic diagnosis of non-malignant lesion; (b) no evidence of HCC in other liver segments; (c) no evidence of extrahepatic metastases; (d) AFP serum level below 400 ng/dl; and (e) absence of informed consent for liver biopsy. Lesions that disappeared or transformed into HCC within the first 6 months followup were excluded. Lesions that transformed into HCC or disappeared after at least 6 months from their first detection were included. For the inclusion of lesions that did not modify their size and/or echopattern, a minimum 18 months follow-up was required. Non-malignant unchanged nodules with a shorter follow up were not analyzed. At entry, biochemical and serologic screening included standard liver function tests, serum AFP, serum iron, ferritin, caeruloplasmin, alpha-1 antitrypsin, hepatitis B, C and delta virus markers. Smooth-muscle antibodies, antinuclear antibodies and antimitocondial antibodies, were determined in each case. Ultrasound scans were performed by using a realtime US scanner (Toshiba SAL-30) with a 3.5-MHz convex transducer.
2.1. Liver histology Intra- and extranodular fine-needle biopsy under US guidance using a 21 gauge cutting needle was carried out in all cases. Specimens were routinely processed and stained by Hematoxylin-eosin, Gomori for reticulin framework, PAS with and without diastase digestion and Masson
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Table 1 Morphologic features adopted in this study for the classification of macroregenerative nodules in cirrhosis
Bulging clonal growth Map-like clonal growth Plate thickening Pseudoglands Unpaired arteries Sinusoidal capillarization Nuclear peripheral alignment Cell crowding Large cell change Small cell change Reticulum framework
LRN
LGDN
HGDN
WDHCC
2 2 2 2 2 þ /2 2 2 2 2 þ
2 þ/2 þ/2 2 þ/2 þ/2 2 2 þ 2 þ
þ/2 þ þ þ/2 þ þ þ/2 þ/2 þ/2 þ þ/2
þ þ þ þ þ þ þ þ 2 þ þ /2
LRN, large regenerative nodule; LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule; and WDHCC, well differentiated hepatocellular carcinoma. trichrome. Each liver sample was independently analyzed by two pathologists (AM.C, M.R.). According to recently proposed criteria [2], macronodules were grouped into three main histologic categories: large regenerative nodule (LRN), low-grade dysplastic (LGDN) and high-grade dysplastic nodule (HGDN). Diagnostic features helping in the differential diagnosis are listed in Table 1. In particular a nodule was classified as large regenerative when no distinctive cyto-architectural features as compared to extralesional sample were observed. The diagnosis of LGDN referred to a nodule characterized by preserved hepatic architecture with two cell thick hepatocyte plates, low grade cytologic atypias, generally large cell change and inconstant detection of map-like clonal growth or increased number of unpaired arterioles (Figs. 1a,b,l). The diagnosis of HGDN was made when two or more of the following architectural abnormalities were present: (1) nuclear crowding; (2) two to three cell thick hepatocyte plates; (3) clonal growth such of map-like and bulging type; and (4) microacini formation. In these nodules an increased number of unpaired arteries is usually detectable. For morphological details of HGDNs see Figs. 1c–e,g– i,m. Histological diagnosis of HCC was based on internationally accepted criteria [9]. Large cell changes (LCC, large cell dysplasia) were diagnosed according to Anthony et al. [10] and small cell changes (small cell dysplasia) according to Watanabe et al. [11].
2.2. Follow-up In all patients clinical evaluation, liver function tests, US scanning and serum AFP determination were repeated every 3 months. Each lesion was subjected to control biopsy if changes in the US pattern and/or size enlargement occurred. Focal liver lesions discovered by US scanning during follow-up were evaluated by fine-needle biopsy to ascertain their benign or malignant nature. HCC occurring either from a preexisting nodule or as ‘de novo’ lesion was confirmed by histology and examined by other imaging techniques including CT either conventional or spiral, MRI and selective hepatic arteriography plus lipiodol injection followed by CT. Nodule disappearance was confirmed in at least two consecutive US examinations taken at 3 months interval.
2.3. Serologic evaluation Serum markers of HBV infection and serum AFP were determined by commercial radioimmunoassay kits. Serum anti-hepatitis C virus (HCV) antibodies were assayed by the ‘Ortho-HCV’ ELISA first and second generation test (Ortho Diagnostics, Raritan, NJ, USA).
2.4. Statistical analysis Differences in means and proportions were evaluated by a Student’s t-test and a chi-squared test, respectively. The rate of HCC incidence was
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(62%), detected during US surveillance, were considered as incident. Their main size was 17.7 ^ 5.7 mm; 83 (92%) had a maximum diameter less than 3 cm with 8 (9%) measuring , 1 cm, 66 (73%) 1– 2 cm, and 9 (10%) 2 – 3 cm. Twentythree nodules (40%) were hyperechoic and hyperecogenicity was a reflection of fat in seven of them (30%). The mean basal AFP value was 12.6 ^ 6.9 ng/ml, being in the normal range in 90% of cases; a single case showed elevated serum AFP values (149 ng/ml). Macronodules were classified as LRN (56 cases, 61%), LGDN (16 cases, 18%) and HGDN (19 cases, 21%). The basal characteristics of macronodules are depicted in Table 2. Given that patients with LRN and LGDN nodules did not differ by sex, age, etiology, mean size, mean AFP and US pattern we compared basal characteristics of LRN and LGDN (group A) with those HGDN (group B). No difference was observed according to mean age, mean basal size, severity of cirrhosis, platelets count, and mean basal AFP values. In contrast, HGDN were more likely detected in males, more likely hyperechoic and more likely associated with extranodular large cell changes (Table 3). Fig. 1. Architectural (a – h) and nuclear (i – m) basic lesions in macronodules. Basic lesions in a, b, l were detected in low grade dysplastic nodules (LGDN); basic lesions in c, d, e, g, h, i and m were detected in high grade dysplastic nodules (HGDN). (a) A focus of two to three cell thick hepatocytes plates (right) as opposed to adjacent one to two cell thick (left) hepatocytes plates. (b) Map-like clonal growth, clear cell type, with indistinct cell boundaries, adjacent to a portal tract. (c) Bulging clonal growth, clear cell type: Notice the compressed adjacent parenchyma (arrows). (d) Bulging clonal growth, clear cell type with small cell change and hepatocyte thickening. (e) A hepatocyte pseudogland (arrow) lacking Kupffer cell rimming. (f) Hepatocyte tubularization near a focal lesion. The pericentral location and intraluminal bile plugs are signs of liver compression. (g) Sinusoidal nuclear alignment (arrows) in association with nuclear crowding. (h) Nuclear crowding (middle and right) with clear cell change as opposed to normal cell density (upper left). (i) A small arteriole unassociated to a portal tract. Large cell change and microacinar structures (arrow) can also be seen. (l) Large cell change in a periportal/periseptal location. (m) Small cell change (middle and left) characterized by increased N/C ratio, cell density and plate thickening. A focus of large cell change (right). calculated by the person-year method. The Kaplan–Meier method [12] was used to estimate the cumulative probability of HCC development during follow-up according to ultrasonographic pattern, presence or absence of large cell change, and presence or absence of histologic diagnosis of highgrade dysplastic nodule. Differences in HCC development were evaluated with a log rank test. A P value ,0.05 was considered significant. Independent predictors of HCC development were evaluated in Cox proportional hazards regression analysis [13]. In the model HCC development was the outcome variable; presence of hyperechoic nodule, presence of large cell change, and histologic diagnosis of high-grade dysplastic nodule, were the independent variables.
3. Results 3.1. Characteristics of macronodules at enrollment Thirty-four macronodules (38%) were prevalent and 56
3.2. Outcome The overall mean follow-up was 33 months (range, 6– 97 months). During this period, 28/90 nodules (31%) transformed into HCC. Cumulative HCC transformation rates at 1, 2, 3, and 5 years were 3.5, 15.5, 31, and 48.5%, respectively. The incidence of HCC per 100 person-years of follow-up was 11.3%. At univariate analysis (Table 4) only three variables were associated with the likelihood of neoplastic evolution, namely the histologic diagnosis of HGDN, the extranodular detection of LCC and the US hyperechoic pattern. Given that subjects with LGDN were Table 2 Basal characteristics of large regenerative (LRN), low-grade (LGDN) and high-grade (HGDN) dysplastic nodules Characteristics
No. Age M/F Child-Pugh A/B/C Bilirubin mg/dl Platelets/ml ( £ 1000) Etiology HCV HBV HCV-HBV Alcohol Others Size (mm) AFP (ng/dl) Hypo/hyperechoic
Nodules LRN
LGDN
HGDN
55 58 (33–77) 34/21 47/8/0 0.9 (0.5–4.7) 98 (65–131)
16 57 (56–71) 11/5 15/1/0 0.8 (0.5–2.8) 95 (68–195)
19 61 (62–74) 16/3 17/2/0 0.9 (0.5–3.9) 94 (56–178)
33 7 1 7 7 16.7 (9–32) 11.5 (2–146) 41/14
3 4 1 4 4 17 (10–30) 7.5 (2–19) 15/1
7 5 1 2 4 16.8 (10–33) 13.3 (4–60) 11/8
Data are expressed as mean (range).
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Table 3 Comparison of basal clinical, biochemical, ultrasound and histological characteristics of macronodules Characteristics
Sex Mean age (years) Child-Pugh
Macronodules
F M A B
Mean bilirubin (mg/dl) Mean platelets ( £ 1000/ml) Etiology HBV HCV HBV-HCV Alcohol Cryptogenic Haemochromatosis PBC Mean basal size (mm) Mean basal AFP (ng/dl) Large cell change (extranodular) Ultrasound hyperechoic
Group A (LRN and LGDN) (no. 71)
Group B (HGDN) (no. 19)
26 45 58 ^ 12 62 9 0.8 ^ 0.3 101 ^ 45 11 36 2 11 6 3 2 17.2 ^ 5.9 12.4 ^ 19.8 24 15
3 16 61 ^ 11 17 2 0.9 ^ 0.4 94 ^ 36 5 7 1 2 3 1 – 16.8 ^ 5.2 13.3 ^ 13.1 12 8
not at increased risk for HCC development (OR 1.2; CI 95% ¼ 0.3 – 4.4) for further statistical analysis subjects with LRN and LGDN were grouped together and compared to those with HGDN. Figs. 2 –4 show the cumulative probability of HCC occurrence by presence or absence of HGDN, hyperechoic nodules, and extranodular LCC. The differences were statistically significant according to the log rank test for each of the considered conditions. Adjusting for the confounding effect of each of these variables in the Cox proportional hazards model we observed that histologic diagnosis of HGDN (hazard risk, 2.4; CI 95% ¼ 1.1– 5.8) and presence of extranodular LCC (hazard risk, 3.1; CI
P value
,0.05 n.s. n.s. n.s. n.s. n.s.
n.s. n.s. ,0.01 ,0.05
95% ¼ 1.2– 7.8) were both independent predictors of the likelihood of HCC development; the hyperechoic pattern of nodules was unassociated (hazard risk, 1.7; CI 95% ¼ 0.7– 4.0) with HCC development (Table 5). We further evaluated the joint effect of HGDN and extranodular LCC on the risk of HCC development. Using as reference category (OR ¼ 1) subjects without these two conditions, the risk of cancer was 3.9-fold higher in subjects with only HGDN, 4.5-fold higher in those with only LCC and 9.1-fold higher in subjects with HGDN and LCC (Table 6). Thus, an additive interaction on the risk of HCC development is observed in the presence of both these conditions.
Table 4 Variables associated with HCC development in cirrhotic patients with macronodules Variable
Transformed nodules (no. 28)
Untrasformed nodules (no. 62)
P value
OR (CI 95%)
Mean age (years) Sex F M Etiology HBV HCV HBV-HCV Alcohol Cryptogenic Haemochromatosis PBC Mean basal size (mm) Mean basal AFP (ng/ml) Large cell change (extranodular) Histological diagnosis LRN LGDN HGDN Ultrasound hyperechoic
61.8 ^ 8.7 8 20 6 13 1 3 3 1 1 18.4 ^ 5.9 17.1 ^ 27.8 18
58 ^ 8.6 21 41 10 30 2 10 6 3 1 16.6 ^ 5.2 10.5 ^ 12.1 18
n.s. n.s.
1.3 (0.5–3.4)
12 4 12 11
43 12 7 12
n.s.
n.s. n.s. ,0.01
5.7 (2.0–16)
,0.01 ,0.05
1 1.2 (0.3–4.4) 6.1 (1.7–22.4) 2.7 (1– 7)
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Fig. 2. Cumulative probability of macronodules to remain HCC-free according to diagnosis (Kaplan–Meier).
Fig. 4. Cumulative probability of macronodules to remain HCC-free according to 6extranodular LCC (Kaplan– Meier).
During follow-up, eight additional HCCs developed outside the original macronodule so that in the whole series, the rate of liver malignant transformation further increased to 40%. Fifteen macronodules (17%) became persistently undetectable at US after a mean interval of 24.8 months (range 6– 85 months). Of these nodules, 13 (87%) were LRN/LGDN and 2 (13%) HGDN. This favorable evolution was more frequently observed in nodules without than in those with extranodular LCC (13/35 versus 2/36, P , 0:03). Additional macronodules were discovered in 11 cases with six patients developing 1, two patients 2 and three patients 3 macronodules, respectively. Two patients had the original macronodule disappeared but developed a new one 10 and 15 months later. When the final analysis was performed, 79 patients were still alive; and 11 dead during the follow-up. Eight deaths occurred in patients who developed HCC, being the tumor the direct cause of death in six of them and the remaining five deaths were due to liver failure.
detected by US during a screening program for HCC in compensated cirrhotic patients. Our data indicate that macronodules in a cirrhotic setting, identify a subpopulation at high risk of HCC. Particularly prone to HCC development are nodules histologically labelled, on small biopsies, as high-grade dysplastic and those associated with extranodular LCC. Most important, when both these two conditions are present, the risk is higher than the arithmetic sum of the risk associated with the presence of each one of the single condition (additive interaction). By contrast, LRNs and LGDNs have a significantly lower risk to transform into HCC because they usually stabilize or disappear over time. Our study confirmed that macronodules arising in cirrhosis, regardless their histologic classification, are per se strong predictors of an increased risk of malignancy. Indeed, the documented HCC incidence rate of 11.3 cases per 100 person-year of follow-up is from three to five times higher than the corresponding figures reported in two previous Italian studies. In fact, in these studies an incidence rate of 2.5 cases per 100 person-years (29 HCC cases among 217 subjects who received a mean follow-up of 33 months) among cirrhotic patients with different etiologies and different Child grades [14] and an incidence rate of 3.7 cases per 100 person-years (51 HCC cases among 259 subjects who received a mean follow-up of 64.5 months) among cirrhotic patients with only viral etiology Child grade A [15] were reported. Moreover, in our series, considering either HCCs occurring from preexisting nodules and de novo lesions, the overall rate of hepatic
4. Discussion In this study we evaluated the natural outcome of a large series of non-neoplastic macronodules consecutively
Table 5 Multivariate analysis of factors associated with HCC development (Cox proportional Hazards model)
Fig. 3. Cumulative probability of hypoechoic and hyperechoic macronodules to remain HCC-free according to ultrasonographic pattern (Kaplan–Meier).
Variable
Hazard risk
95% CI
High grade dysplastic nodule Extranodular large cell changes Hyperechoic nodule
2.4 3.1 1.7
1.1 –5.8 1.2 –7.8 0.7 –4.0
Each variable is adjusted for the confounding effect of all other listed in the table.
M. Borzio et al. / Journal of Hepatology 39 (2003) 208–214 Table 6 Risk of HCC development in subjects with ^HGDN and ^LCC HGDN
LCC
O.R. (CI 95%)
Absent Present Absent Present
Absent Absent Present Present
1 4.5 (0.9–24.1) 3.9 (1.9–11.5) 9.1 (2.9–29.1)
malignant transformation was 40%, an impressive finding given the relatively short follow up. Interestingly, only 31% of the original nodules transformed into HCC while the majority stabilized or disappeared. These findings outline the need to adequately classify every macronodule detected by US. It is our opinion that the best approach to these lesions is to evaluate their histology in order to select lesions at very high risk of HCC transformation. Furthermore, this study provided evidence that in cirrhosis, every nodule detected by US cannot be assumed to be malignant so that any effort should be made to distinguish true HCC from non-malignant lesions. This behaviour is likely to have a great impact particularly on the management of patient with multiple nodules being therapeutic decision (ranging from surgery to OLT) strictly dependent on their number and histologic characterization [1]. To classify our lesions we followed recently proposed morphologic criteria, not yet clinically validated [2]. These criteria were proposed on the basis of the recognition of certain histologic features in macronodules detected in explanted or autoptic livers. However little is known on the morphological features and changes of these lesions during chronic liver disease, when the natural history of cirrhosis is far to be concluded. We cannot exclude that the available criteria may be not fully exhaustive if applied to ‘in vivo’ series, thus requiring further revision in the near future. Nevertheless our data undoubtedly indicate that, among macronodules, those we labelled HGDNs are truly precancerous, because 63% of them transformed into HCC during follow-up. Our data are in keeping with those recently reported by Terasaky et al. who, in a Japanese series, showed a significantly increased risk of malignant transformation in nodules fulfilling the histological criteria for HGDN [16]. Thus, the correct labelling of a nodule as high-grade dysplastic is of great clinic significance for its predictive adverse value. The finding that, in our as well as in other series, not all HGDNs transformed into HCC does not contrast with the natural history of genuine precancerous lesions, needing sequential additional molecular steps in their transition to malignancy [5,7,8,16]. This may be dependant on a number of variables being the time and the oncogenic background the most important. In the present study two HGDNs disappeared. This unexpected outcome was already reported by Seky et al. in their small series of dysplastic nodules, even though, in that study, low and high-grade DNs were
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grouped together, a feature that may account for the reported high rate (45%) of nodule disappearance [7]. These results, although still acceptable in biologic terms because complete regression can even occur in malignant tumours, may raise some concerns on the reliability of histology on small liver fragments. We accept that certain subtle histologic features can be missed or, alternatively, overestimated in fine needle microbiopsies, leading to nodule misclassification. Unfortunately, to date, no others diagnostic tools including imaging techniques as MRI, spiral TC have proved to be able to discriminate among macronodules [17,18]. More recently, contrast-enhanced doppler ultrasonography has been reported to provide a better characterization of the pattern of vascularization of non-malignant nodules arising in cirrhosis, with HGDN having a low resistance arterial flow, but these findings need to be confirmed in larger series [19]. It is also possible that in a given nodule the combination of size above 1.5 – 2 cm and the vascular profile could have helped to set the diagnosis or predict the risk of malignancy. Unfortunately in the present series this hypothesis could not be tested because only a small proportion of the nodules, namely those discovered more recently, were investigated by imaging techniques as spiral TC or contrast-enhanced doppler ultrasonography. The low sensitivity of imaging is mainly due to the lack of overt derangement in the vascular profile of these lesions. Indeed a diagnostic enhancement at spiral TC is typically found in well-differentiated HCCs and begins to appear in more mature dysplastic nodules. Conversely, in earlier dysplastic nodules, even in the presence of consistent morphologic atypias, neovascularization is still insufficient for enabling a clear-cut imaging diagnosis [20,21]. Immunohistochemical investigations targeted to evaluate the vascular setting of nodules or to document their proliferative rate may also be of additional value [23]. Genetic informations are expected to provide useful diagnostic clues [24] but the molecular events of the human hepatic carcinogenesis occurring in cirrhosis are far from being established. Our results cast some doubts on the clinical significance of LGDNs as opposed to LRNs, at least when considering the time-period of our follow up. At variance with the latter, LGDNs are morphologically characterized by mild atypias including large cell change. As a group these nodules seem to behave as the large regenerative ones, sharing a lower propensity for malignant transformation rate (25%). Their distinction from LRNs on small liver samples is often difficult also because LCC, as found by other authors [22], is detectable in most of them either inside and outside the nodule so that intra and extralesional samples, by showing overlapping features, also fulfill criteria for a diagnosis of LRNs [2]. Given their similar malignant potential, the distinction between LRNs and LGDN in in vivo studies, is unlike to be clinically meaningful and we propose to manage these lesions as a single group. The most fruitful clinical approach seems to strictly follow-up them by US and to repeat sampling in dubious cases. Our study confirmed the role of LCC as strong
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morphologic predictor of HCC appearance. In our patients the presence of extranodular LCC was an independent variable correlated with the malignant transformation, carrying a 3-fold increased risk. The prognostic significance of LCC was already documented in several prospective and retrospective studies [24 – 27]. In these studies the risk of HCC in patients with LCC was 4 – 6-fold increased. However, from the analysis of the survival curves from these studies, the time elapsing between the diagnosis of LCC and HCC occurrence was several years long and the rate of HCC development at 3 years interval did not exceed 50%. In the present study, patients with cirrhosis and LCC and macronodules, regardless their classification, showed a 75% chance of developing an HCC within the first 3 years of observation. In addition, the strict correlation observed in our series between LCC and HGDN indicates that large changes are a particularly favourable background for the emergence of dysplastic nodules and their progression towards malignancy. In conclusion, the present study indicates that macronodule appearance in a cirrhotic setting identifies a subgroup of patients carrying a high risk of HCC development. The risk is particularly striking in patients with HGDN associated to extranodular LCC and therefore, these patients should be strictly monitored and possibly candidated to OLT. Our results support the notion that HGDN are HCC precursors and as such these lesions should be correctly identified by histology and perhaps, preventively treated. Conversely, LGDN and LRN are prevalent lesions characterized by a lower malignant risk. Although for these lesions the optimal follow-up has not yet been established, we think adequate a 3 months US surveillance.
References [1] Bruix J, Llovet MJ. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology 2002;35:519 –524. [2] International Working Party, Terminology of nodular lesions of the liver. Hepatology 1995;22:983–993. [3] Theise N. Macroregenerative (Dysplastic) nodules and hepatocarcinogenesis: theoretical and clinical consideration. Sem Liver Dis 1995; 15:360–371. [4] Kondo F, Ebara M, Sugiura N, Wada K, Kita K, Hirooka N, et al. Histological features and clinical course of large regenerative nodules: evaluation of their precancerous potentiality. Hepatology 1990;12:592 –598. [5] Takayama T, Makuuchi M, Hirohashi S, Sakamoto M, Okazaki N, Takayasu K, et al. Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 1990;336:1150–1153. [6] Rapaccini L, Pompili M, Caturelli E, Anti M, Aliotta A, Cedrone A, et al. Focal ultrasound lesions in liver cirrhosis diagnosed as regenerating nodules by fine-needle biopsy. Follow-up of 12 cases. Dig Dis Sci 1990;35:422–427. [7] Seki S, Sakaguchi H, Kitada T, Tamori A, Takeda T, Kawada N, et al. Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study. Clin Cancer Res 2000;6:3469–3473. [8] Borzio M, Borzio F, Croce A, Sala M, Salmi A, Leandro G, et al.
[9]
[10] [11]
[12] [13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Ultrasonography-detected macroregenerative nodules in cirrhosis: a prospective study. Gastroenterology 1997;112:1617–1623. Gibson JB, Sobin LH. Histological typing of tumors of the liver, biliary tract and pancreas. International histological classification of tumors, no. 20. Geneva: World Health Organization; 1978. p. 12 –30. Anthony PP, Vogel CL, Barker LF. Liver cell dysplasia: a premalignant condition. J Clin Pathol 1973;26:217–223. Watanabe S, Okita K, Harada T, Kodama T, Numa Y, Takemoto T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;51: 2197–2205. Kaplan EL, Meier P. Non-parametric estimation for incomplete observation. J Am Stat Assoc 1958;53:457– 481. Cox DR. Regression models and life tables. J R Stat Soc 1972;34: 187 –220. Colombo M, De Franchis R, Del Ninno E, Sangiovanni A, De Fazio C, Tommasini M, et al. Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med 1991;325:675 –680. Chiaramonte M, Stroffolini T, Vian A, Stazi MA, Floreani A, Lorenzoni U, et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999;85:2132–2137. Terasaki S, Kanedo S, Kobayashi K, Nonamoura A, Nakanuma Y. Histological features predicting malignant transformation of nonmalignant hepatocellular nodules: a prospective study. Gastroenterology 1998;115:1216–1222. Earls JP, Theise ND, Weinreb JC, DeCorato DR, Krinsky GA, Rofsky NM, et al. Dysplastic nodules and hepatocellular carcinoma: thinsection MR imaging of explanted cirrhotic livers with pathologic correlation. Radiology 1996;201:207–214. Lim JH, Cho JM, Kim EY, Park CK. Dysplastic nodules in liver cirrhosis: evaluation of hemodynamics with CT during arterial portography and CT hepatic arteriography. Radiology 2000;214:869–874. Fracanzani AL, Burdick L, Borzio M, Roncalli M, Bonelli N, Borzio F, et al. Contrast-enhanced doppler ultrasonography in the diagnosis of hepatocellular carcinoma and premalignant lesions in patients with cirrhosis. Hepatology 2001;34:1109– 1112. Park YN, Yang CP, Fernandez GJ, Cubukcu O, Thung S, Theise N. Neoangiogenesis and sinusoidal capillarization in dysplastic nodules of the liver. Am J Surg Pathol 1998;22:656 –662. 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: implication for diagnosis and classification. Hepatology 1999;30:1174– 1178. Le Bail B, Bellanne`e G, Bernard P-H, Saric J, Balabaud C, BioulacSage P. Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 macronodular lesions in cirrhotic explants of 10 adult French patients. Hum Pathol 1995;26:897–906. Maggioni M, Coggi G, Cassani B, Bianchi P, Romagnoli S, Mandelli A, et al. Molecular changes in hepatocellular dysplastic nodules on microdissected liver biopsies. Hepatology 2000;32:942 –946. Borzio M, Bruno S, Roncalli M, Colloredo Mels G, Ramella G, Borzio F, et al. Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: a prospective study. Gastroenterology 1995;108:812 –817. Ganne-Carrie´ N, Chastang C, Chapel F, Munz C, Pateron D, Sibony M, et al. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis. Hepatology 1996;23:1112– 1118. Lee RG, Tsamandas AC, Demetris AJ. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched casecontrol study, pathological analysis, and pathogenetic hypothesis. Hepatology 1997;26:1415 –1422. Fracanzani AL, Borzio M, Roncalli M, Derenzini M, Trere` D, Mattioli M, et al. Can large cell change and high proliferative activity predict hepatocellular carcinoma in patients with hereditary hemochromatosis. Am J Gastroenterol 2000;95:2940–2945.
Impact of large regenerative, low grade and high grade dysplastic nodules in hepatocellular carcinoma development Mauro Borzio1,2,*, Silvia Fargion3, Franco Borzio1,2, Anna Ludovica Fracanzani3, Anna Maria Croce1,2, Tommaso Stroffolini4, Silvia Oldani1,2, Rodolfo Cotichini5, Massimo Roncalli6 1
Department of Medicine, Gastroenterology Unit, Ospedale Fatebenefratelli, C.so Porta Nuova 23, 20121 Milan, Italy Department of Pathology, Gastroenterology Unit, Ospedale Fatebenefratelli, C.so Porta Nuova 23, 20121 Milan, Italy 3 Department of Internal Medicine, Ospedale Maggiore IRCCS, Universita` di Milano, Milan, Italy 4 Liver Unit, Ospedale San Giacomo, Rome, Italy 5 Laboratory of Epidemiology, Istituto Superiore di Sanita`, Rome, Italy 6 Department of Pathology, Istituto Clinico Humanitas, Rozzano, Universita` di Milano, Milan, Italy
2
See Editorial, pages 269 – 271
Background/Aims: The natural outcome of ultrasound-detected macronodules in cirrhosis is still poorly understood. In this study we assessed the incidence and predictors of malignant transformation in a prospective study of 90 consecutive ultrasound-detected macronodules in cirrhosis. Methods: Macronodules classification was based on recently proposed histological criteria. Extranodular large (LCC) and small cell changes were also evaluated. The follow-up included ultrasound and serum alfa-fetoprotein determination every 3 months. Independent predictors of hepatocellular carcinoma were evaluated by Cox proportional hazards regression analysis. Results: During a mean follow-up of 33 months, 28 (31%) nodules transformed into hepatocellular carcinoma. The incidence of hepatocellular carcinoma per 100 person-years of follow-up was 11.3%, with a malignant transformation rate of 3.5, 15.5, 31 and 48.5% at 1, 2, 3, and 5 years respectively. High-grade dysplastic nodules (HGDN) (hazard risk 5 2.4; CI 95% 5 1.1– 5.0) and LCC (hazard risk 5 3.1; CI 95% 5 1.2 –7.8) were independent predictors of malignant transformation. Eight additional hepatocellular carcinomas developed outside the original lesions raising the overall malignant transformation rate to 40% while 15 macronodules (17%) became undetectable at ultrasound (US). Conclusions: Macronodules characterize a cirrhotic subpopulation with high risk of hepatocellular carcinoma. HGDN and LCC are strong predictors of malignant transformation; subjects with simultaneous presence of both these two conditions are at highest risk of cancer development. The management of cirrhotics with macronodules should be based on morphologic features detected on liver microsamples. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Large regenerative nodule; Low-grade dysplastic nodule; High-grade dysplastic nodule hepatocellular carcinoma; Cirrhosis
1. Introduction Ultrasound detection of small nodular lesions in cirrhosis is becoming a routine daily practice, requiring an accurate Received 2 December 2002; received in revised form 27 March 2003; accepted 9 April 2003 * Corresponding author. Tel.: þ39-2-6363-2596; fax: þ 39-2-6363-2665. E-mail address: [email protected] (M. Borzio).
diagnostic workout. The correct labeling of any hepatic nodule and the number of those truly malignant are essential for a proper clinical management, particularly when surgery and orthotopic liver transplantation (OLT) are available options, given the stringent inclusion criteria recently proposed [1]. The differential diagnosis of small nodular lesions arising in cirrhosis is basically restricted to early hepatocellular carcinoma (HCC) and non-malignant macronodules which
0168-8278/03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-8278(03)00190-9
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include large regenerative, low and high grade dysplastic nodules [2]. To date, the prevailing opinion is that high grade dysplastic nodules are the true HCC precursors while the remaining nodules are though to be only marginally involved in human liver carcinogenesis [3]. Nevertheless, the clinical significance of macronodules detected ‘in vivo’ and their management are still debated and under the focus of extensive research. Indeed, most of the studies addressing their natural outcome were retrospective or mainly restricted to large regenerative or atypical/border-line lesions [4 – 7]. In a recent study of 32 ultrasound (US)-detected hepatocellular nodules, some variables predicting their malignant transformation have emerged but, given the small size of the cohort, results were considered preliminary [8]. In the present study we definitely report on the natural outcome of a large series of macronodules consecutively detected by US, during a program of HCC surveillance in cirrhosis, histologically classified according to the criteria proposed by the International Working Party [2]. The aim of this study was to ascertain the truly malignant potential of the different hepatic macronodules and to establish the predictive significance of additional clinic, histologic and radiologic variables.
2. Materials and methods This study was carried out on 90 consecutive patients (61 male and 29 female) with US-detected macronodules identified at two different Hospitals from 1990 to 1998. Patients were part of a larger series of more than 400 cirrhotics recruited in a screening program for HCC surveillance undergoing US and alfa-fetoprotein (AFP) determination every 6 months. Only patients fulfilling inclusion criteria (see below) were admitted to this study. All focal liver lesions detected during follow-up were histologically examined by an intra and extranodular sampling under US guidance. In case of first negative/unclear biopsy a second biopsy was repeated within 3 months. The histologic diagnosis was also supported by conventional or spiral computed tomography (CT) or MRI or hepatic arteriography plus lipiodol infusion. None of the patients enrolled in this study were treated by interferon after inclusion. The following inclusion criteria were used: (a) histologic diagnosis of non-malignant lesion; (b) no evidence of HCC in other liver segments; (c) no evidence of extrahepatic metastases; (d) AFP serum level below 400 ng/dl; and (e) absence of informed consent for liver biopsy. Lesions that disappeared or transformed into HCC within the first 6 months followup were excluded. Lesions that transformed into HCC or disappeared after at least 6 months from their first detection were included. For the inclusion of lesions that did not modify their size and/or echopattern, a minimum 18 months follow-up was required. Non-malignant unchanged nodules with a shorter follow up were not analyzed. At entry, biochemical and serologic screening included standard liver function tests, serum AFP, serum iron, ferritin, caeruloplasmin, alpha-1 antitrypsin, hepatitis B, C and delta virus markers. Smooth-muscle antibodies, antinuclear antibodies and antimitocondial antibodies, were determined in each case. Ultrasound scans were performed by using a realtime US scanner (Toshiba SAL-30) with a 3.5-MHz convex transducer.
2.1. Liver histology Intra- and extranodular fine-needle biopsy under US guidance using a 21 gauge cutting needle was carried out in all cases. Specimens were routinely processed and stained by Hematoxylin-eosin, Gomori for reticulin framework, PAS with and without diastase digestion and Masson
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Table 1 Morphologic features adopted in this study for the classification of macroregenerative nodules in cirrhosis
Bulging clonal growth Map-like clonal growth Plate thickening Pseudoglands Unpaired arteries Sinusoidal capillarization Nuclear peripheral alignment Cell crowding Large cell change Small cell change Reticulum framework
LRN
LGDN
HGDN
WDHCC
2 2 2 2 2 þ /2 2 2 2 2 þ
2 þ/2 þ/2 2 þ/2 þ/2 2 2 þ 2 þ
þ/2 þ þ þ/2 þ þ þ/2 þ/2 þ/2 þ þ/2
þ þ þ þ þ þ þ þ 2 þ þ /2
LRN, large regenerative nodule; LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule; and WDHCC, well differentiated hepatocellular carcinoma. trichrome. Each liver sample was independently analyzed by two pathologists (AM.C, M.R.). According to recently proposed criteria [2], macronodules were grouped into three main histologic categories: large regenerative nodule (LRN), low-grade dysplastic (LGDN) and high-grade dysplastic nodule (HGDN). Diagnostic features helping in the differential diagnosis are listed in Table 1. In particular a nodule was classified as large regenerative when no distinctive cyto-architectural features as compared to extralesional sample were observed. The diagnosis of LGDN referred to a nodule characterized by preserved hepatic architecture with two cell thick hepatocyte plates, low grade cytologic atypias, generally large cell change and inconstant detection of map-like clonal growth or increased number of unpaired arterioles (Figs. 1a,b,l). The diagnosis of HGDN was made when two or more of the following architectural abnormalities were present: (1) nuclear crowding; (2) two to three cell thick hepatocyte plates; (3) clonal growth such of map-like and bulging type; and (4) microacini formation. In these nodules an increased number of unpaired arteries is usually detectable. For morphological details of HGDNs see Figs. 1c–e,g– i,m. Histological diagnosis of HCC was based on internationally accepted criteria [9]. Large cell changes (LCC, large cell dysplasia) were diagnosed according to Anthony et al. [10] and small cell changes (small cell dysplasia) according to Watanabe et al. [11].
2.2. Follow-up In all patients clinical evaluation, liver function tests, US scanning and serum AFP determination were repeated every 3 months. Each lesion was subjected to control biopsy if changes in the US pattern and/or size enlargement occurred. Focal liver lesions discovered by US scanning during follow-up were evaluated by fine-needle biopsy to ascertain their benign or malignant nature. HCC occurring either from a preexisting nodule or as ‘de novo’ lesion was confirmed by histology and examined by other imaging techniques including CT either conventional or spiral, MRI and selective hepatic arteriography plus lipiodol injection followed by CT. Nodule disappearance was confirmed in at least two consecutive US examinations taken at 3 months interval.
2.3. Serologic evaluation Serum markers of HBV infection and serum AFP were determined by commercial radioimmunoassay kits. Serum anti-hepatitis C virus (HCV) antibodies were assayed by the ‘Ortho-HCV’ ELISA first and second generation test (Ortho Diagnostics, Raritan, NJ, USA).
2.4. Statistical analysis Differences in means and proportions were evaluated by a Student’s t-test and a chi-squared test, respectively. The rate of HCC incidence was
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(62%), detected during US surveillance, were considered as incident. Their main size was 17.7 ^ 5.7 mm; 83 (92%) had a maximum diameter less than 3 cm with 8 (9%) measuring , 1 cm, 66 (73%) 1– 2 cm, and 9 (10%) 2 – 3 cm. Twentythree nodules (40%) were hyperechoic and hyperecogenicity was a reflection of fat in seven of them (30%). The mean basal AFP value was 12.6 ^ 6.9 ng/ml, being in the normal range in 90% of cases; a single case showed elevated serum AFP values (149 ng/ml). Macronodules were classified as LRN (56 cases, 61%), LGDN (16 cases, 18%) and HGDN (19 cases, 21%). The basal characteristics of macronodules are depicted in Table 2. Given that patients with LRN and LGDN nodules did not differ by sex, age, etiology, mean size, mean AFP and US pattern we compared basal characteristics of LRN and LGDN (group A) with those HGDN (group B). No difference was observed according to mean age, mean basal size, severity of cirrhosis, platelets count, and mean basal AFP values. In contrast, HGDN were more likely detected in males, more likely hyperechoic and more likely associated with extranodular large cell changes (Table 3). Fig. 1. Architectural (a – h) and nuclear (i – m) basic lesions in macronodules. Basic lesions in a, b, l were detected in low grade dysplastic nodules (LGDN); basic lesions in c, d, e, g, h, i and m were detected in high grade dysplastic nodules (HGDN). (a) A focus of two to three cell thick hepatocytes plates (right) as opposed to adjacent one to two cell thick (left) hepatocytes plates. (b) Map-like clonal growth, clear cell type, with indistinct cell boundaries, adjacent to a portal tract. (c) Bulging clonal growth, clear cell type: Notice the compressed adjacent parenchyma (arrows). (d) Bulging clonal growth, clear cell type with small cell change and hepatocyte thickening. (e) A hepatocyte pseudogland (arrow) lacking Kupffer cell rimming. (f) Hepatocyte tubularization near a focal lesion. The pericentral location and intraluminal bile plugs are signs of liver compression. (g) Sinusoidal nuclear alignment (arrows) in association with nuclear crowding. (h) Nuclear crowding (middle and right) with clear cell change as opposed to normal cell density (upper left). (i) A small arteriole unassociated to a portal tract. Large cell change and microacinar structures (arrow) can also be seen. (l) Large cell change in a periportal/periseptal location. (m) Small cell change (middle and left) characterized by increased N/C ratio, cell density and plate thickening. A focus of large cell change (right). calculated by the person-year method. The Kaplan–Meier method [12] was used to estimate the cumulative probability of HCC development during follow-up according to ultrasonographic pattern, presence or absence of large cell change, and presence or absence of histologic diagnosis of highgrade dysplastic nodule. Differences in HCC development were evaluated with a log rank test. A P value ,0.05 was considered significant. Independent predictors of HCC development were evaluated in Cox proportional hazards regression analysis [13]. In the model HCC development was the outcome variable; presence of hyperechoic nodule, presence of large cell change, and histologic diagnosis of high-grade dysplastic nodule, were the independent variables.
3. Results 3.1. Characteristics of macronodules at enrollment Thirty-four macronodules (38%) were prevalent and 56
3.2. Outcome The overall mean follow-up was 33 months (range, 6– 97 months). During this period, 28/90 nodules (31%) transformed into HCC. Cumulative HCC transformation rates at 1, 2, 3, and 5 years were 3.5, 15.5, 31, and 48.5%, respectively. The incidence of HCC per 100 person-years of follow-up was 11.3%. At univariate analysis (Table 4) only three variables were associated with the likelihood of neoplastic evolution, namely the histologic diagnosis of HGDN, the extranodular detection of LCC and the US hyperechoic pattern. Given that subjects with LGDN were Table 2 Basal characteristics of large regenerative (LRN), low-grade (LGDN) and high-grade (HGDN) dysplastic nodules Characteristics
No. Age M/F Child-Pugh A/B/C Bilirubin mg/dl Platelets/ml ( £ 1000) Etiology HCV HBV HCV-HBV Alcohol Others Size (mm) AFP (ng/dl) Hypo/hyperechoic
Nodules LRN
LGDN
HGDN
55 58 (33–77) 34/21 47/8/0 0.9 (0.5–4.7) 98 (65–131)
16 57 (56–71) 11/5 15/1/0 0.8 (0.5–2.8) 95 (68–195)
19 61 (62–74) 16/3 17/2/0 0.9 (0.5–3.9) 94 (56–178)
33 7 1 7 7 16.7 (9–32) 11.5 (2–146) 41/14
3 4 1 4 4 17 (10–30) 7.5 (2–19) 15/1
7 5 1 2 4 16.8 (10–33) 13.3 (4–60) 11/8
Data are expressed as mean (range).
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Table 3 Comparison of basal clinical, biochemical, ultrasound and histological characteristics of macronodules Characteristics
Sex Mean age (years) Child-Pugh
Macronodules
F M A B
Mean bilirubin (mg/dl) Mean platelets ( £ 1000/ml) Etiology HBV HCV HBV-HCV Alcohol Cryptogenic Haemochromatosis PBC Mean basal size (mm) Mean basal AFP (ng/dl) Large cell change (extranodular) Ultrasound hyperechoic
Group A (LRN and LGDN) (no. 71)
Group B (HGDN) (no. 19)
26 45 58 ^ 12 62 9 0.8 ^ 0.3 101 ^ 45 11 36 2 11 6 3 2 17.2 ^ 5.9 12.4 ^ 19.8 24 15
3 16 61 ^ 11 17 2 0.9 ^ 0.4 94 ^ 36 5 7 1 2 3 1 – 16.8 ^ 5.2 13.3 ^ 13.1 12 8
not at increased risk for HCC development (OR 1.2; CI 95% ¼ 0.3 – 4.4) for further statistical analysis subjects with LRN and LGDN were grouped together and compared to those with HGDN. Figs. 2 –4 show the cumulative probability of HCC occurrence by presence or absence of HGDN, hyperechoic nodules, and extranodular LCC. The differences were statistically significant according to the log rank test for each of the considered conditions. Adjusting for the confounding effect of each of these variables in the Cox proportional hazards model we observed that histologic diagnosis of HGDN (hazard risk, 2.4; CI 95% ¼ 1.1– 5.8) and presence of extranodular LCC (hazard risk, 3.1; CI
P value
,0.05 n.s. n.s. n.s. n.s. n.s.
n.s. n.s. ,0.01 ,0.05
95% ¼ 1.2– 7.8) were both independent predictors of the likelihood of HCC development; the hyperechoic pattern of nodules was unassociated (hazard risk, 1.7; CI 95% ¼ 0.7– 4.0) with HCC development (Table 5). We further evaluated the joint effect of HGDN and extranodular LCC on the risk of HCC development. Using as reference category (OR ¼ 1) subjects without these two conditions, the risk of cancer was 3.9-fold higher in subjects with only HGDN, 4.5-fold higher in those with only LCC and 9.1-fold higher in subjects with HGDN and LCC (Table 6). Thus, an additive interaction on the risk of HCC development is observed in the presence of both these conditions.
Table 4 Variables associated with HCC development in cirrhotic patients with macronodules Variable
Transformed nodules (no. 28)
Untrasformed nodules (no. 62)
P value
OR (CI 95%)
Mean age (years) Sex F M Etiology HBV HCV HBV-HCV Alcohol Cryptogenic Haemochromatosis PBC Mean basal size (mm) Mean basal AFP (ng/ml) Large cell change (extranodular) Histological diagnosis LRN LGDN HGDN Ultrasound hyperechoic
61.8 ^ 8.7 8 20 6 13 1 3 3 1 1 18.4 ^ 5.9 17.1 ^ 27.8 18
58 ^ 8.6 21 41 10 30 2 10 6 3 1 16.6 ^ 5.2 10.5 ^ 12.1 18
n.s. n.s.
1.3 (0.5–3.4)
12 4 12 11
43 12 7 12
n.s.
n.s. n.s. ,0.01
5.7 (2.0–16)
,0.01 ,0.05
1 1.2 (0.3–4.4) 6.1 (1.7–22.4) 2.7 (1– 7)
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Fig. 2. Cumulative probability of macronodules to remain HCC-free according to diagnosis (Kaplan–Meier).
Fig. 4. Cumulative probability of macronodules to remain HCC-free according to 6extranodular LCC (Kaplan– Meier).
During follow-up, eight additional HCCs developed outside the original macronodule so that in the whole series, the rate of liver malignant transformation further increased to 40%. Fifteen macronodules (17%) became persistently undetectable at US after a mean interval of 24.8 months (range 6– 85 months). Of these nodules, 13 (87%) were LRN/LGDN and 2 (13%) HGDN. This favorable evolution was more frequently observed in nodules without than in those with extranodular LCC (13/35 versus 2/36, P , 0:03). Additional macronodules were discovered in 11 cases with six patients developing 1, two patients 2 and three patients 3 macronodules, respectively. Two patients had the original macronodule disappeared but developed a new one 10 and 15 months later. When the final analysis was performed, 79 patients were still alive; and 11 dead during the follow-up. Eight deaths occurred in patients who developed HCC, being the tumor the direct cause of death in six of them and the remaining five deaths were due to liver failure.
detected by US during a screening program for HCC in compensated cirrhotic patients. Our data indicate that macronodules in a cirrhotic setting, identify a subpopulation at high risk of HCC. Particularly prone to HCC development are nodules histologically labelled, on small biopsies, as high-grade dysplastic and those associated with extranodular LCC. Most important, when both these two conditions are present, the risk is higher than the arithmetic sum of the risk associated with the presence of each one of the single condition (additive interaction). By contrast, LRNs and LGDNs have a significantly lower risk to transform into HCC because they usually stabilize or disappear over time. Our study confirmed that macronodules arising in cirrhosis, regardless their histologic classification, are per se strong predictors of an increased risk of malignancy. Indeed, the documented HCC incidence rate of 11.3 cases per 100 person-year of follow-up is from three to five times higher than the corresponding figures reported in two previous Italian studies. In fact, in these studies an incidence rate of 2.5 cases per 100 person-years (29 HCC cases among 217 subjects who received a mean follow-up of 33 months) among cirrhotic patients with different etiologies and different Child grades [14] and an incidence rate of 3.7 cases per 100 person-years (51 HCC cases among 259 subjects who received a mean follow-up of 64.5 months) among cirrhotic patients with only viral etiology Child grade A [15] were reported. Moreover, in our series, considering either HCCs occurring from preexisting nodules and de novo lesions, the overall rate of hepatic
4. Discussion In this study we evaluated the natural outcome of a large series of non-neoplastic macronodules consecutively
Table 5 Multivariate analysis of factors associated with HCC development (Cox proportional Hazards model)
Fig. 3. Cumulative probability of hypoechoic and hyperechoic macronodules to remain HCC-free according to ultrasonographic pattern (Kaplan–Meier).
Variable
Hazard risk
95% CI
High grade dysplastic nodule Extranodular large cell changes Hyperechoic nodule
2.4 3.1 1.7
1.1 –5.8 1.2 –7.8 0.7 –4.0
Each variable is adjusted for the confounding effect of all other listed in the table.
M. Borzio et al. / Journal of Hepatology 39 (2003) 208–214 Table 6 Risk of HCC development in subjects with ^HGDN and ^LCC HGDN
LCC
O.R. (CI 95%)
Absent Present Absent Present
Absent Absent Present Present
1 4.5 (0.9–24.1) 3.9 (1.9–11.5) 9.1 (2.9–29.1)
malignant transformation was 40%, an impressive finding given the relatively short follow up. Interestingly, only 31% of the original nodules transformed into HCC while the majority stabilized or disappeared. These findings outline the need to adequately classify every macronodule detected by US. It is our opinion that the best approach to these lesions is to evaluate their histology in order to select lesions at very high risk of HCC transformation. Furthermore, this study provided evidence that in cirrhosis, every nodule detected by US cannot be assumed to be malignant so that any effort should be made to distinguish true HCC from non-malignant lesions. This behaviour is likely to have a great impact particularly on the management of patient with multiple nodules being therapeutic decision (ranging from surgery to OLT) strictly dependent on their number and histologic characterization [1]. To classify our lesions we followed recently proposed morphologic criteria, not yet clinically validated [2]. These criteria were proposed on the basis of the recognition of certain histologic features in macronodules detected in explanted or autoptic livers. However little is known on the morphological features and changes of these lesions during chronic liver disease, when the natural history of cirrhosis is far to be concluded. We cannot exclude that the available criteria may be not fully exhaustive if applied to ‘in vivo’ series, thus requiring further revision in the near future. Nevertheless our data undoubtedly indicate that, among macronodules, those we labelled HGDNs are truly precancerous, because 63% of them transformed into HCC during follow-up. Our data are in keeping with those recently reported by Terasaky et al. who, in a Japanese series, showed a significantly increased risk of malignant transformation in nodules fulfilling the histological criteria for HGDN [16]. Thus, the correct labelling of a nodule as high-grade dysplastic is of great clinic significance for its predictive adverse value. The finding that, in our as well as in other series, not all HGDNs transformed into HCC does not contrast with the natural history of genuine precancerous lesions, needing sequential additional molecular steps in their transition to malignancy [5,7,8,16]. This may be dependant on a number of variables being the time and the oncogenic background the most important. In the present study two HGDNs disappeared. This unexpected outcome was already reported by Seky et al. in their small series of dysplastic nodules, even though, in that study, low and high-grade DNs were
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grouped together, a feature that may account for the reported high rate (45%) of nodule disappearance [7]. These results, although still acceptable in biologic terms because complete regression can even occur in malignant tumours, may raise some concerns on the reliability of histology on small liver fragments. We accept that certain subtle histologic features can be missed or, alternatively, overestimated in fine needle microbiopsies, leading to nodule misclassification. Unfortunately, to date, no others diagnostic tools including imaging techniques as MRI, spiral TC have proved to be able to discriminate among macronodules [17,18]. More recently, contrast-enhanced doppler ultrasonography has been reported to provide a better characterization of the pattern of vascularization of non-malignant nodules arising in cirrhosis, with HGDN having a low resistance arterial flow, but these findings need to be confirmed in larger series [19]. It is also possible that in a given nodule the combination of size above 1.5 – 2 cm and the vascular profile could have helped to set the diagnosis or predict the risk of malignancy. Unfortunately in the present series this hypothesis could not be tested because only a small proportion of the nodules, namely those discovered more recently, were investigated by imaging techniques as spiral TC or contrast-enhanced doppler ultrasonography. The low sensitivity of imaging is mainly due to the lack of overt derangement in the vascular profile of these lesions. Indeed a diagnostic enhancement at spiral TC is typically found in well-differentiated HCCs and begins to appear in more mature dysplastic nodules. Conversely, in earlier dysplastic nodules, even in the presence of consistent morphologic atypias, neovascularization is still insufficient for enabling a clear-cut imaging diagnosis [20,21]. Immunohistochemical investigations targeted to evaluate the vascular setting of nodules or to document their proliferative rate may also be of additional value [23]. Genetic informations are expected to provide useful diagnostic clues [24] but the molecular events of the human hepatic carcinogenesis occurring in cirrhosis are far from being established. Our results cast some doubts on the clinical significance of LGDNs as opposed to LRNs, at least when considering the time-period of our follow up. At variance with the latter, LGDNs are morphologically characterized by mild atypias including large cell change. As a group these nodules seem to behave as the large regenerative ones, sharing a lower propensity for malignant transformation rate (25%). Their distinction from LRNs on small liver samples is often difficult also because LCC, as found by other authors [22], is detectable in most of them either inside and outside the nodule so that intra and extralesional samples, by showing overlapping features, also fulfill criteria for a diagnosis of LRNs [2]. Given their similar malignant potential, the distinction between LRNs and LGDN in in vivo studies, is unlike to be clinically meaningful and we propose to manage these lesions as a single group. The most fruitful clinical approach seems to strictly follow-up them by US and to repeat sampling in dubious cases. Our study confirmed the role of LCC as strong
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morphologic predictor of HCC appearance. In our patients the presence of extranodular LCC was an independent variable correlated with the malignant transformation, carrying a 3-fold increased risk. The prognostic significance of LCC was already documented in several prospective and retrospective studies [24 – 27]. In these studies the risk of HCC in patients with LCC was 4 – 6-fold increased. However, from the analysis of the survival curves from these studies, the time elapsing between the diagnosis of LCC and HCC occurrence was several years long and the rate of HCC development at 3 years interval did not exceed 50%. In the present study, patients with cirrhosis and LCC and macronodules, regardless their classification, showed a 75% chance of developing an HCC within the first 3 years of observation. In addition, the strict correlation observed in our series between LCC and HGDN indicates that large changes are a particularly favourable background for the emergence of dysplastic nodules and their progression towards malignancy. In conclusion, the present study indicates that macronodule appearance in a cirrhotic setting identifies a subgroup of patients carrying a high risk of HCC development. The risk is particularly striking in patients with HGDN associated to extranodular LCC and therefore, these patients should be strictly monitored and possibly candidated to OLT. Our results support the notion that HGDN are HCC precursors and as such these lesions should be correctly identified by histology and perhaps, preventively treated. Conversely, LGDN and LRN are prevalent lesions characterized by a lower malignant risk. Although for these lesions the optimal follow-up has not yet been established, we think adequate a 3 months US surveillance.
References [1] Bruix J, Llovet MJ. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology 2002;35:519 –524. [2] International Working Party, Terminology of nodular lesions of the liver. Hepatology 1995;22:983–993. [3] Theise N. Macroregenerative (Dysplastic) nodules and hepatocarcinogenesis: theoretical and clinical consideration. Sem Liver Dis 1995; 15:360–371. [4] Kondo F, Ebara M, Sugiura N, Wada K, Kita K, Hirooka N, et al. Histological features and clinical course of large regenerative nodules: evaluation of their precancerous potentiality. Hepatology 1990;12:592 –598. [5] Takayama T, Makuuchi M, Hirohashi S, Sakamoto M, Okazaki N, Takayasu K, et al. Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 1990;336:1150–1153. [6] Rapaccini L, Pompili M, Caturelli E, Anti M, Aliotta A, Cedrone A, et al. Focal ultrasound lesions in liver cirrhosis diagnosed as regenerating nodules by fine-needle biopsy. Follow-up of 12 cases. Dig Dis Sci 1990;35:422–427. [7] Seki S, Sakaguchi H, Kitada T, Tamori A, Takeda T, Kawada N, et al. Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study. Clin Cancer Res 2000;6:3469–3473. [8] Borzio M, Borzio F, Croce A, Sala M, Salmi A, Leandro G, et al.
[9]
[10] [11]
[12] [13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Ultrasonography-detected macroregenerative nodules in cirrhosis: a prospective study. Gastroenterology 1997;112:1617–1623. Gibson JB, Sobin LH. Histological typing of tumors of the liver, biliary tract and pancreas. International histological classification of tumors, no. 20. Geneva: World Health Organization; 1978. p. 12 –30. Anthony PP, Vogel CL, Barker LF. Liver cell dysplasia: a premalignant condition. J Clin Pathol 1973;26:217–223. Watanabe S, Okita K, Harada T, Kodama T, Numa Y, Takemoto T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;51: 2197–2205. Kaplan EL, Meier P. Non-parametric estimation for incomplete observation. J Am Stat Assoc 1958;53:457– 481. Cox DR. Regression models and life tables. J R Stat Soc 1972;34: 187 –220. Colombo M, De Franchis R, Del Ninno E, Sangiovanni A, De Fazio C, Tommasini M, et al. Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med 1991;325:675 –680. Chiaramonte M, Stroffolini T, Vian A, Stazi MA, Floreani A, Lorenzoni U, et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999;85:2132–2137. Terasaki S, Kanedo S, Kobayashi K, Nonamoura A, Nakanuma Y. Histological features predicting malignant transformation of nonmalignant hepatocellular nodules: a prospective study. Gastroenterology 1998;115:1216–1222. Earls JP, Theise ND, Weinreb JC, DeCorato DR, Krinsky GA, Rofsky NM, et al. Dysplastic nodules and hepatocellular carcinoma: thinsection MR imaging of explanted cirrhotic livers with pathologic correlation. Radiology 1996;201:207–214. Lim JH, Cho JM, Kim EY, Park CK. Dysplastic nodules in liver cirrhosis: evaluation of hemodynamics with CT during arterial portography and CT hepatic arteriography. Radiology 2000;214:869–874. Fracanzani AL, Burdick L, Borzio M, Roncalli M, Bonelli N, Borzio F, et al. Contrast-enhanced doppler ultrasonography in the diagnosis of hepatocellular carcinoma and premalignant lesions in patients with cirrhosis. Hepatology 2001;34:1109– 1112. Park YN, Yang CP, Fernandez GJ, Cubukcu O, Thung S, Theise N. Neoangiogenesis and sinusoidal capillarization in dysplastic nodules of the liver. Am J Surg Pathol 1998;22:656 –662. 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: implication for diagnosis and classification. Hepatology 1999;30:1174– 1178. Le Bail B, Bellanne`e G, Bernard P-H, Saric J, Balabaud C, BioulacSage P. Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 macronodular lesions in cirrhotic explants of 10 adult French patients. Hum Pathol 1995;26:897–906. Maggioni M, Coggi G, Cassani B, Bianchi P, Romagnoli S, Mandelli A, et al. Molecular changes in hepatocellular dysplastic nodules on microdissected liver biopsies. Hepatology 2000;32:942 –946. Borzio M, Bruno S, Roncalli M, Colloredo Mels G, Ramella G, Borzio F, et al. Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: a prospective study. Gastroenterology 1995;108:812 –817. Ganne-Carrie´ N, Chastang C, Chapel F, Munz C, Pateron D, Sibony M, et al. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis. Hepatology 1996;23:1112– 1118. Lee RG, Tsamandas AC, Demetris AJ. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched casecontrol study, pathological analysis, and pathogenetic hypothesis. Hepatology 1997;26:1415 –1422. Fracanzani AL, Borzio M, Roncalli M, Derenzini M, Trere` D, Mattioli M, et al. Can large cell change and high proliferative activity predict hepatocellular carcinoma in patients with hereditary hemochromatosis. Am J Gastroenterol 2000;95:2940–2945.