Incidence and Diagnostic Features of Macroregenerative Nodules vs. Small Hepatocellular Carcinoma in Cirrhotic Livers LINDAFERRELL,'TERESAWRIGHT,' JOHN LAKE,' JOHN ROBERTS3 AND NANCY &CHER3 Departments of 'Pathology, 2Medicine and "Surgery, University of California, San Francisco, California 94143
In Japan, the presence of a large regenerative nodule within a cirrhotic liver, referred to as a macroregenerative nodule or adenomatous hyperplasia,is thought to play a role in the pathogenesis of hepatocellular carcinoma. These lesions, however, have received little attention outside of Japan. We examined 110 sequentially explanted cirrhotic livers for the presence of such nodules. By gross examination, 19 livers (17.3%) had 40 nodules (10 livers had more than one nodule) between 0.8 and 3.6 cm in diameter. By histological examination, 28 of these were macroregenerative nodules and 12 were hepatocellular carcinomas.Three of these hepatocellular carcinomas, however, appeared to have arisen in association with a macroregenerative nodule. We found that the architectural features of thickened cell plates, formation of trabeculae and loss of reticulin were usually very helpful in differentiating benign macroregenerative nodules from hepatocellular carcinoma.The incidence of macroregenerative nodules in our serieswas similar to that seen in the Japanese studies, and although we feel that they may play a role in the pathogenesis of carcinoma, we do not believe their presence is necessary for the development of hepatocellular carcinoma. (HEPAM M G Y 1992;16:1372-1381.)
confirm malignancy (8).In addition, the Japanese have found MRNs to contain foci of well-differentiated HCC (4,9-11)and have thus come to consider MRNs to be risk factors in the pathogenesis of HCC (1,4, 5, 8, 11). Few investigations, however, have studied the incidence of MRNs and their role in the pathogenesis of HCC in a population without the predominant HBV infection. In this study, we examined 110 sequential liver explants with cirrhoses of various causes to establish the incidence of MRNs and to examine their differentiation from and association with HCC.
MATERIALS AND METHODS We serially sectioned 110 consecutive cirrhotic liver explant specimensat 0.5-cm to 1.0-cmintervals and examined them for the presence of nodules measuring 0.8 to 3.5 cm in greatest diameter (Fig. 1).We used 0.8 cm as the minimum acceptable size, observingthe example of Terada, Hoso and Nakanuma (5) and Terada et al. (9) rather than the l-cm minimum used by Furuya et al. (4) because we found that, on gross examination, nodules of this size stood out significantly against the background liver in micronodular or mixed nodular cirrhoses. In macronodular cirrhoses with very large nodules, this cutoff point may not be valid, and one might only sample nodules of size that had a different color or shape than the other A high incidence of small HCC has been noted in this nodules rather than all nodules. In our cases, this was not an association with cases of HBV infection in Japan (1). issue. Samples from each explant, and every nodule found, Investigators there have used radiographic techniques were fixed in formalin; embedded in paraffin; sectioned at to screen patients with HBV in efforts to identify these 5 km; stained with hematoxylin and eosin, Snook's reticulin small HCCs for early therapy (2). Early therapy reg- and Perl's iron stains; and examined by light microscopy by imens such as hepatectomy, embolization and percuta- L. Ferrell. Each cirrhotic liver was categorized as to origin neous ethanol injection (3) for small lesions have (based on pathological findings, clinical information or both); nodule size (>go% of nodules < 3 mm = micro; 50% of resulted in markedly improved survival rates. nodules > 3 mm = macro; or mixed nodular); iron deposition; In an attempt to make such early diagnoses, Japanese investigators have noted the presence of large regener- and the presence of small-cell or large-cell change of hepato(so-called dysplasia). Small-cell change was defined as ative nodules within cirrhotic livers, so-called macrore- cytes clusters of smaller-than-normal hepatocytes with increased generative nodules (MRNs)or adenomatous hyperplasia nuclearkytoplasmic ratios (Fig. 2A); large-cell change was (1-71, to be a common finding. These benign nodules defined as larger-than-normal hepatocytes with enlarged and cannot be distinguished from malignant nodules by irregular nuclei (Fig. 2B).The extent of small-cell or large-cell radiography, so a histological diagnosis is necessary to change was subjectively graded on a scale of 1 to 3, with 1 = rare, 2 = easily found and 3 = abundant and prominent. The presence of Mallory hyaline and fat was also noted. Each nodule was examined with hematoxylin and eosin h i v e d January 6, 1992; accepted July 27, 1992. stain for the presence of portal tracts or bile ducts; Mallory Address reprint requests to: Linda Ferrell, Department of Pathology, hyaline; small, large, or clear cell change; bile; fat; infiltrative University of California Medical Center, San Francisco, CA 94143-0102. or ill-defined borders; multilobularity; irregular or dilated 31/1/41572 1372
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FIG 1 Explanted liver with mixed nodular cirrhosis and inarrorcgenerati\e nodule The nodule was dark green compared with the more yellow-green background liver
FIG.2. Size differences exist in the hepatocytes in both panels although the photos are the same magnification. (A) Small-cell change (center1 x 325 ). 1 B 1 Large-cell change Note enlarged hepatocytes with large, usually irregularlyshaped nuclei as compared with an adjacent normal-sized hepatocyte ~(irroii'i(1-1 & E. original magnification x. 325).
(H& E, original magnification
sinusoids; and intranodular fibrosis. A nodule was classified as simple MRN if the architectural features were regenerative in nature with a fairly normal reticulin pattern, which, for the most part, was indistinguishable from the background liver (Fig. 3). A nodule was designated at-ypical (borderline HCC or questionable low-grade HCC) if the architectural features were distorted by foci of irregularly thickened cell plates (greater than two cells thick) (Fig. 4) with an associated loss of reticulin. suggesting an abnormal focus ( 4 ) . A thickened cell plate was defined as a small isolated focus of thickening of one or two plates in the same location, suggesting an abnormal prolifer-
ation, but limited in size and orientation as an ill-defined process. A nodule with a definitive trabecular (Fig. 51, pseudoglandular (Figs. 6 and i'),compact or scirrhous pattern was classified as HCC, according to the World Health Organization classification ( 12).Trabeculae were specifically defined in this study as zones of cell plates greater than two cells thick involving several plates in the same area, leading to a pronounced difference in architecture from that of surrounding normal or regenerative plates. Small-cell and large-cell dysplasia-like changes were noted separately and did not influence the classification of nodules (We did not use the
1374
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FERRELL ET AL.
HEPATOLOGY
FIG.3. Macroregenerativenodule, simple type, with uniform cell plate architecturetypical of a cirrhotic nodule (H& E, original magnification 125).
FIG.4. Atypical macroregenerativenodule with irregular, ill-definedcell plates and small-cell change. The focal granularity of the cells is due to iron (H & E, original magnification x 165).
type I and type I1 designations for MRNs [4J or the HCC
A reticulin stain of each nodule was examined for the absence grading system developed by Japanese investigators [l, 81 of or decrease in staining intensity compared with the because we were not trained in the criteria for such grading surrounding cirrhotic liver (10) (Fig. l l ) , for the presence of and we do not necessarily agree with their nomenclature of thickenedcell plates (greater than two hepatocytes in width) or these lesions.) Infiltrating or irregular edges were defined as any other architectural abnormalities such as the formation of the direct extension of a proliferative focus within the nodule microacini (or pseudoglands) or fragmented trabeculae lined into another portion of the nodule or adjacent tissue (Fig. 8). by endothelial cells (10,13). Iron stain was examined on a scale The intranodular fibrosis consisted of fibrous septa, usually of 1 to 4 + for the quantity of iron deposits in the nodule thin, that separated cell plates, and were not associated with a compared with that present in the background liver. portal tract, bile duct or vessels (10) (Fig. 9), but could result Fisher’s exact test (14) was used to test for significant in an appearance of multilobularity within the nodule (Fig. 10). associations regarding HCV infection, presence of MRNs and
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FIG.5 . (A) HCC with broad trabecular lined by entiothelial cells. small-cell change and wide sinusoidal spaces. ( H & E, original magmfication 325). ( B ) Reticulin stain of same lesion shows t h r ahsence ( i f lincvw itaining at the edges of trabeculae. typical of HCC (reticulin, original magnification 325 I . x
presence of HCC. One-sided p values were used because of the ambiguity in defining the second tail for this test ( 151. Because of the small numbers Of atuYPicalMRNs and "CCS, an exact method (16)was also used to test for trends i n the proportions of nodules exhibiting various histological features. HCV infections were confirmed by the polymerase chain reaction previously described I 171. Patients with alcoholic and cryptogenic cirrhosis were confirmed to he negative for HCV by the polymerase chain reaction. All patients had a minimum of 5 mo clinical follow-up
RESULTS The orisns for the cirrhoses, the size of the and the extent of smdl-cell Or lmge-ceu change found in each explant are listed in Tables 1-3. Forty nodules rangmg in diameters from 0.8 to 3.5 cm from l 9 patients (17.3% of 110 patients) were found (Fig. 1 ) . The number of nodules Per Patient, their dlstribution and type (simple, atypical or HCC) and hackground liver findings in each patient are listed in
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FERRELLETAL
HEPATOLOGY
FIG.7. Formation of pseudoglands, or microacini (urrowsl, with small-cell change in a liver 3 wk after transplantation. Most of the other hepatocytes are of normal size (H & E, original magnification x 325).
FIG.8. Infiltrative edge of HCC (center),with well-differentiated HCC on right and adjacent nodule on left. Note the small clusters of atypical cells as seen in the HCC infiltrating the fibrous stroma outside of the main nodule mass (H & E, original magnification x 63).
Table 4. Twenty-five nodules found in 12 patients were classified as simple, 6 nodules in 3 patients were classified as atypical and 9 nodules in 5 patients were classified as HCCs only. In addition, three nodules contained both HCC and a residual rim of tissue with architectural and cytological features of MRN (one simple, two atypical) for a total of 12 HCCs in eight patients. Four patients had both MRN and HCC, and patients with at least one MRN had a much higher rate of HCC (4 of 15, 26.3%) than those with no MRNs
(4 of 95, 4.2%,p = 0.012). One of the three patients with atypical nodules and four of the eight patients with HCC had cirrhosis caused by HCV. Thus having HCV as the cause of the liver disease was associated with an increased chance of having a nodule of any type (30%vs. 12.5%for all others, p = 0.034) and of having HCC (13% vs. 5%, p = 0.14). The incidence of HCC diagnosed as this size of nodule was 12 of 43 (28%), with the end result that 8 of 19 patients (42%) with nodules (7% of the 110 total cases reviewed) received
Vol. 16, No. 6, 1992
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FIG. 9. Intranodular fibrosis of H(’( Thr thln fibrous septa separate trabeculae of the tumor and do not contmn bile ducts or other portal structures This HCC also shows fattc c hmge rH & E: oriplnal magnlficatlon 6 3 1
a malignant diagnosis. Eleven of the 19 patients (58%; 10% of the total cases reviewed) received a benign diagnosis of MRN only. Pathological features of the MRNs and HCCs are listed in Table 5 . Portal tracts or isolated bile ducts were not present in HCCs. Mallory hyaline, clear cell change and bile were seen in both types of MRNs and in malignant lesions. All six atypical nodules had small-cell change, five had foci of decreased reticulin and four had foci of thickened cell plates, but all three features were
never seen in the same focus in a given nodule. The lack of or marked decrease in reticulin (Figs. 5 and 111, lack of iron and trabeculae formation were the only features seen in all of the HCCs; however, many of the benign nodules lacked iron also. We considered the definitive formation of trabeculae in the nodule as the best diagnostic criterion (or definition) for carcinoma. In doing so, we noted an attendant loss of reticulin framework in the malignant nodules (p < 0.001). An infiltrative border or multilobularity and intranodular
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FERRELL ET AL.
HEPATOLOGY
FIG.11. Core biopsy specimen of cirrhotic liver with well-differentiated HCC, reticulin stain, diagnosis confirmed on explant tissue. (A) Reticulin of the background liver is well demonstrated and distinct, and the cell plates of the cirrhotic nodule do not fall apart (reticulin, original magnification x 125). (B) In contrast, the HCC has essentially no reticulin framework, and consequently breaks into smaller, dissociated cell plates, many only two to three cells in thickness, in this field. Other fields had foci of fragmented trabecular plates up to five cells thick. Note the small-cell change (reticulin, original magnification x 1251.
TABLE 1. Origin of cirrhotic explants AU livers (n =
Livers with nodules (n = 19P
110)
Origin
Number
Percentage
Hepatitis C virus Alcohol Cryptogenic Autoimmune Sclerosing cholangitis Biliary atresia HBV PBC Hemochromatosis
30 25 19 7 10 5
27 23 17 6 9 4
5
4
7 2
6 2
Number
Percentage
~
47 (30% of hepatitis C virus) 11 (8% of alcohol) 26 (26% of cryptogenic) 5 (14% of autoimmune)
5 (14% of PBC) 5 (50% of hemochromatosis)
"Nodules defined as measuring 0.8 to 3.5 cm.
TABLE 2. Type of background cirrhosis AU livers (n =
110) Livers with nodules (n
Description Number Percentage Number ~~
~~
Macronodular Micronodular Mixednodular
4 28
78
=
19)"
Percentage
~~
4 25 71
2
3 14
10 (50% of macro) 16 (11% of micro) 74 (18% of mixed)
"Nodules defined as measuring 0.8 to 3.5 cm.
unremarkable MRNs (Fig. 2A). Small-cell change was also prevalent in the areas of trabeculae or pseudoglands in the HCCs (Fig. 6A). So-called large-cell dysplasia was a common finding (Fig. 2B).In our study, 65 (59%)of the examined livers, including 14 (74%)with nodules (MRNs and HCCs) and 5 (62%)with HCC, had some degree of large cell change. Twenty-six (60%) of the nodules showed large-cell change, including 4 of 6 atypical MRNs (67%) with large-cell change and 8 of 12 (67%) of the HCCs.
DISCUSSION fibrosis were also significantly more typical of HCC In our study, the incidence of patients who had (p < 0.001). We were able to identify clusters of small-cell change nodules that ranged in size from 0.8 to 3.5 cm in in 52% (13 of 25) of simple MRNs, 100% (6 of 6) of diameter was 17.3%; 7% (eight patients) had diagnoses atypical MRNs and 75% (9 of 12) of the HCCs. Single or of HCC and the remaining 10% proved to be MRNs. Two multiple clusters could be present in otherwise totally studies from Japan showed an incidence of MRNs of
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TABLE 3. Cytological changes in background cirrhosis All livers
CyLOlogical atypia
1.
Small-cell change Large-cell change
-13
7
22
:34
2-
Livers with nodulesf
3 .
Total (percentage of 110 livers)
1+
24
3 4-
0 9
14 11:?%I
4
2
0
65 (60%I
2
8
4
Total (percentage of 18)
6 (32%) 14 ( 73%)'
1 + = rare, 2 I- = moderate number5 .1 ahundant "Nodules defined as measuring 0 8 ti) 3 5 cm *Expressed as number of livers 'Five of eight livers w t h HCC included 4
TABLE 4. Type of nodules and relationship to origin and type of cirrhosis Number of patients
Number of noduledpatient
Morphological type
Origin of cirrhosis
Size of cirrhotic nodules
4
1
S
3 mixed, 1 micro
3 HCV, CC
3 3 1 1 1 1 1
)
S
i
HC"
ti 1
S
2 mixed, micro Mixed Macro Mixed Macro Mixed Mixed Mixed Mixed Mixed Mixed
HCV, ETOH, PBC CC, HCV, ETOH Autoimmune H CV C3C HCV HCV
1
1 1 1
s S
:i 1
> > I 1
2A. I-ICt' in A s. 4 €lC(k A. k1CC i n A 2 II(Ts HCC' in S A
c:c
HCV
(XI Hema
S = simple macroregenerative nodule; A at-ypical macroregenerative nodule; Macro = macronodular; Mixed Micro = micronodular; CC = c~yptogeniccirrhosis: ETOH alcohol: Herno = hemochromatosis.
14.2% (49 of 345 cases [41, with nodules measuring greater than 1 cm), and 37% (53 of 141 cases [61, with nodules measuring greater than 0.5 cm); thus the incidence of these lesions in our study was comparable to those seen in Japan despite the fact that our population had a different etiological pattern. Most patients in our study had HCV (27%) or alcoholic (23% or cryptogenic types of cirrhosis ( 16%1. The patients in the Japanese studies. on the other hand, showed a predominance of HBV. In Furuya et al.'s study (4), 38.8% of the patients with livers examined for MRNs ( a total of 345) were positive for HBsAg and 32 of the 49 MRNs (65%) found were in livers that also contained foci of HCC. The exact number of HBV-positive patients with both an MRN and a focus of HCC was not stated, but the authors did state that 49% of patients with an MRN (with or without associated HCC) were HBV positive. In contrast, in our study only 6 of 43 (14%) of the MRNs were found in livers with foci of HCC. Only 4% (five patients) were HBV positive, and none of our HBV patients had an MRN or a focus of HCC. This difference may suggest that the comparatively lower incidence of HCC associated with the MRNs in our study may in part be caused by the absence of HBV (18-21) in these livers. Additional studies with more cases may be necessary to confirm this relationship. Another possible explanation could be the use of dif-
=
mixed nodular;
ferent criteria for the diagnosis of carcinoma in Japan than in our study, which may result in a lower incidence of malignancy. It is interesting to note that four of the eight patients with HCC in our study were HCV positive, and two of these patients had MRNs. In addition, 9 of the 19 patients with nodules were HCV positive (47%),whereas only 27% of the total number of patients were HCV positive. Alcohol, on the other hand, was the cause for cirrhosis in 23% of the patients, but only two of these This prevalence of MRN patients had a nodule (11%). and HCC in the HCV group may suggest a similar association of HCV with these lesions as seen in the HBV population. Evidence suggesting that HCV may play a causal role in the development of HCC has been reported (22). Various histopathological features of MRNs and small HCCs were examined in the Japanese studies, and it was shown that HCCs have an increased incidence compared with simple MRNs if so-called small-cell change (4, 8, 23), lack of iron deposits (9, 111, trabeculae (1, 101, pseudoglands or microacini (1, 8, 101, peritrabecular fibrosis (10) and loss of reticulin framework occurred ( 10). MRNs and HCCs may both show clusters of clear cells that contain Mallory bodies (5,24-26) and increased amounts of bile (10). Our study supports some but not all of these findings (Table 5). Our main difficulty was in distinguishing small-cell
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FERRELL ET AL.
TABLE 5. Pathological features of MRNs and small HCCs
Portal tractsbile ducts Lack of ironb Mallory hyaline Small-cell change Large-cell change Increased bile Clear-cell change, focal Clear-cell change, diffuse Fatty change Irregular sinusoids or peliosis Decreased reticulin' Infiltrating borders or multilobular areas of atypia' Intranodular fibrosisd Plates more than two cells thick
HCC
Simple (n = 26)
Atypical (n = 6)
14 15 14 13 14 9
0 12 4 9
5
3 4 1 6 4 1 4
3 7
0.002 0.019 0.170 0.150 0.600 0.470 0.019
0
0
2
0.073
4
0 2
6 2
0.043 1.00
5
11 9
< 0.001
6 12
< 0.001 < 0.001
4 3 0
0 0
1
1 4
(n = 12)
8
pValue"
< 0.001
MRN = macroregenerative nodule; Simple = nodules without architectural abnormalities, resembling routine regenerative nodules; Atypical = nodules with focal architectural abnormalities including broader cell plates consistent with a proliferative focus within the nodule. "Test for either increasing or decreasing proportions, with atypical considered to be midway between simple and HCC. The p values for the comparison of atypical vs. HCC for the last three features are 0.032, 0.20 and 0.10, respectively. *No iron or decreased over the background cirrhotic liver. 'Edge of nodule shows focal irregularity, extending beyond the confines of the border of the lesion in an infiltrative manner or has the appearance of a multilobular lesion. dFibrosis not associated with a portal tract or bile duct, consisting of fibrous septa separating cell plates.
dysplasia from regenerative changes with small hepatocytes or compression of hepatocytes by regenerative foci
(Figs. 2, 5). At times, small cells would appear to be compressed by adjacent regenerating tissue and at other times would seem to be admixed with a nodular regenerative focus. We also found a rare pseudogland or microacini in these foci. We recognize that Kondo et al. (8) have proposed that lesions with small-cell dysplasia and microacini should be classified as foci of HCC even in the presence of normal cell plates, but we do not necessarily agree that this should be the minimal malignant criterion. We have seen numerous examples in transplanted livers of regenerative foci with small cells and microacini that were histologically similar to the described foci of small-cell dysplasia (Fig. 7). These findings are especially prominent the first few weeks &er transplant, when the liver is actively regenerating to replace cells lost during transport and perfusion. We suggest that many of these small foci in MRNs may represent similar regenerative processes and not necessarily a malignant process. On the other hand, we agree that larger zones of small-cell change should be ex-
HEPATOLOGY
amined closely for the underlying architectural abnormalities associated with the trabecular, pseudoglandular or compact types of HCC because small-cell change can be a typical feature of HCC (Fig. 5 ) . Large-cell change (Fig. 2B) was also not a discriminatory finding because it was seen abundantly in the MRNs and in the cirrhotic livers with and without nodules. Thus the presence of large-cell change as an isolated finding is not a diagnostic marker for distinguishing cirrhosis from MRN or HCC. In addition, our findings do not confirm a definitive preneoplastic role for large-cell dysplasia, and one might suggest that at least some of the large atypical cells represent a form of regenerative or even degenerative change. Of the remaining histological features of MRNs and HCCs (Table 51, the loss or absence of reticulin, thickened cell plates (more than two cells), intranodular fibrous septa and infiltrative borders or multilobular areas of atypia may be the features that would most raise the suspicion for HCC because of the high prevalence of these findings in the HCCs but not in the simple type of MRNs. Whether an atypical lesion that shows some of these features should be considered low-grade HCC is debatable. Standardization of criteria for the classification of such atypical nodules would help in this regard. The presence of multiple large nodules was also a confusing factor. A multifocal process could represent multifocal HCC, as was the case in two patients. Three of our patients, however, had one or more nodules of HCC in addition to one or more MRNs, and six patients had more than one MRN. Because lesions of this size and/or multiplicity could potentially be discovered with radiographic techniques (2), pathologists should be aware of the relatively high frequency of benign nodules and be prepared to differentiate MRNs from HCCs. In addition, in these instances of multifocality, one might be faced with the dilemma of which nodule to sample or remove because the pathological finding of any one nodule may not be representative of the pathological finding of the other nodules. A problem that will most likely arise in the diagnosis of such nodules will be the size and type of sample obtained. Because of the relatively small size of the nodules, many biopsy specimens are likely to contain surrounding cirrhotic tissue. Fine-needle aspiration biopsy (27,28)with smear examination alone may not be sufficient to make the diagnosis because the presence of large-cell or small-cell dysplasia within an MRN or the surrounding liver parenchyma could be misinterpreted as HCC, especially on less cellular specimens. A cell button of a fine-needleaspiration biopsy sample or a core biopsy sample allows for better examination of the architectural features (e.g., trabeculae formation and loss of reticulin) (Fig. 11) that can distinguish benign lesions from well-differentiated HCCs. A core biopsy specimen might provide a better sample than a cell button, but, as with a fine-needle aspiration biopsy sample, it is imperative to obtain representative material to avoid sampling error. Thus the pathologist will
MA(’K( )KEGENERATIVE NODVLES VS HC‘C’
Vol 16. No 6, 1992
no doubt need to communicate with the gastroenterologist and radiologist to ensure adequate sampling of the region in question. In summary, we conclude that MRNs are relatively common findings outside of Japan and that these benign lesions may be more easily separated histologically from well-differentiated HCC by their architectural features in conjunction with the cytological features rather than by cytology alone. We support the issue that the MRNs may themselves predispose to the formation of HCC, but they do not represent the only predisposing factor because our study contained several small HCCs without associated MRNs. We also suggest that cofactors such as HCV may enhance the risk for HCC with or without the presence of an underlying MRN.
ri
9
10
11
12
Acknowledgments: We thank David Geller for editorial advice and assistance and Peter Bacchitti (from the Liver Center, University of California. San Fran- 1 1 cisco, supported by Liver Core Center Grant AM-26743 1 for the statistical analysis. We also wish to acknowledge 14 the International Liver Pathology Study Group, consisting of the following members: C. Balabaud, Bordeaux 15 Cedex, France; K. Bardadin, Warsaw. Poland; P. 16 Bioulac-Sage, Bordeaux Cedex, France; J. Crawford, Boston, MA; R. Colombari, Verona, Italy: P. Dhillon, London, UK; L. Ferrell, San Francisco. CA; Y. Na- 17 kanuma, Kanazawa, Japan; B. Portmann. London. UK; J. Rode, Melbourne, Australia; P. Scheuer. London, LJK; D. Snover, Minneapolis, MN; N . Theise, New York, NY; 18 S. Thung, New York, NY; W. Tsui, Kowloon, Hong Kong; and D. VanLeeuwen. Amsterdam, The Nether- 19 lands. This group discussed the issue of MRNs at their meeting in London in July 1990 (generously supported 20 by Ortho Diagnostic, Dako and Roche Products) and provided valuable input and inspiration for this study. REFERENCES 1. Sakamoto M, Hirohashi S. Shimosato Y. Early stages of’multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol 1991:22:172-178. 2. Rapaccini G , Pornpili M. Caturelli E.Anti M. Aliottu A, Cedrone A. Amadei E, 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. 3. Yamasaki S, Hasegawa H, Makuuchi M , l’adatoshi ‘I‘. Kosuge ‘1’. Shimada K. Choice of treatments for sinall hepatocellular carcinoma: hepatectomy, embolization. or ethanol injection. J Gastroenterol Hepatol 1991:6:408-413 4. Furuya K, Nakamurd M. Yamamoto Y. ‘I’ogei K, Otsuka H. Macroregenerative nodule of the liver. a clinicopat hologic study of 345 autopsy cases of chronic liver disease. Cancer 1988;61:99-105. 5. Terada T, Hoso M, Nakanuma Y. Mallor?. body clustering in adenomatous hyperplasia in human cirrhotic livers: report of four cases. Hum Pathol 1989;20:886-89(1. 6. Wada K, Kondo F, Kondo Y. Large regenerative nodules and dysplastic nodules in cirrhotic livers. a histopathologic study. HEPATOLKY1988;8:1684- 1688. 7 . Kondo F, EbaraM, Sugiura N, Wada K. f i t a K. Hirooka N, Nagoto Y, et al. Histological features and clinical course of large regener-
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ative nodules: evaluation of their precancerous potentiality. HF:P.~TC)I.(H;Y 1990;12:592-598. Kondo F, Wada K, Nagato Y, Nakajima T, Kondo Y, Hirooka N, Ebara M. et al. Biopsy diagnosis of well-differentiated hepatocellular carcinoma based on new morphologic criteria. HEPATOLWY 1989;9:751-755. Terada ‘1’, Kadoya M, Nakanuma Y, Matusui 0. Iron-accumulating adenomatous hyperplastic nodule with malignant foci in the cirrhotic liver: histopathologic, quantitative iron, and magnetic resonance imaging in uitro studies. Cancer 1990;65:1994-2000. Nakanuma Y. Ohta G, Sugiura H, Watanabe K, Doishita K. Incidental solitary hepatocellular carcinomas smaller than 1 cm in size found at autopsy: a morphologic study. HEPATOLOCY 1986;6: 631-635. ’I’erada T, Nakanuma Y. Survey of iron-accumulative macroregenerative nodules in cirrhotic livers. HEPATOLOGY 1989;10: 85 1-854. Gibson JB. Sobin LH. eds. Histolog~altyping of tumours of the liver, biliary tract and pancreas. In: International histological classification of tumours, No. 20. Geneva: World Health Organization. 1978:20-22. Wakasa K. Sakurai M, Okamura J , Kuroda C. Pathological study of small hepatocellular carcinoma: frequency of their invasion. Virchows Arch IA] 1985;407:259-270. Schlesselman JJ. Case control studies: design, conduct, analysis. Oxford: Oxford University Press, 1982:180. Davis I d Exact tests for 2 x 2 contingency tables. Am Stat 1986;40:139- 141. Statistics and Epidemiology Research Corporation. EGRET reference manual. Seattle: Statistics and Epidemiology Research Corporation, 1990:183-189. Cristiano K, Di Bisceglie A, Hoofnagle J. Feinstone S. Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by polymerase chain reaction using multiple primer sets. HEPATOLOGY 199 1 ;14:51-55 Beasley RP. Hepatitis B virus: the major etiology of hepatocellular carcinoma. Cancer 1988;61:1942-1956. Akagi G . Furuya K, Kanamura A, Chihara T, Otsuka H. Liver cell dysplasia and hepatitis B surface antigen in liver cirrhosis and hepatocellular carcinoma. Cancer 1984;54:315-318. Lok A. Lai CL. Factors determining the development of hepatocellular carcinoma in hepatitis B surface antigen carriers: a comparison between families with clusters and solitary cases. Cancer 1988;61:1287-1291. Leuschner 1, Harms D. Schmidt D. The association of hepatocellular carcinoma in childhood with hepatitis B virus infection. Cancer 1988;62:2363-2369. IGyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. HEPATOLOGY 1990;12: 671-675. Crawford J . Pathologic assessment of liver cell dysplasia and benign liver tumors: differentiation from malignant tumors. Semin Diagn Pathol 1990;7:115-128. Nakanuma Y, Ohta G. Expression of Mallory bodies in hepatoeellular carcinoma in man and its significance. Cancer 1986;57:81-86. Nakanuma Y, Ohta G . Is Mallory body formation a preneoplastic change: a study of 181 cases of liver bearing hepatocellular carcinoma and 82 cases of cirrhosis. Cancer 1985;55:2400-2404. Nakanuma Y, Ohta G . Small hepatocellular carcinoma containing many Mallory bodies. Liver 1984;4:128-133. Cohen M, Haber M, Holly E, Ahn D, Bottles K, Stoloff A. Cytologic criteria to distinguish hepatocellular carcinoma from nonneoplastic liver. Am J Clin Pathol 1991;95:125-130, Tao L, Ho C, McLoughlin M, Evans W, Donat E. Cytologic diagnosis of hepatocellular carcinoma by fine-needle aspiration biopsy. Cancer 1984;53:547-552.
In Japan, the presence of a large regenerative nodule within a cirrhotic liver, referred to as a macroregenerative nodule or adenomatous hyperplasia,is thought to play a role in the pathogenesis of hepatocellular carcinoma. These lesions, however, have received little attention outside of Japan. We examined 110 sequentially explanted cirrhotic livers for the presence of such nodules. By gross examination, 19 livers (17.3%) had 40 nodules (10 livers had more than one nodule) between 0.8 and 3.6 cm in diameter. By histological examination, 28 of these were macroregenerative nodules and 12 were hepatocellular carcinomas.Three of these hepatocellular carcinomas, however, appeared to have arisen in association with a macroregenerative nodule. We found that the architectural features of thickened cell plates, formation of trabeculae and loss of reticulin were usually very helpful in differentiating benign macroregenerative nodules from hepatocellular carcinoma.The incidence of macroregenerative nodules in our serieswas similar to that seen in the Japanese studies, and although we feel that they may play a role in the pathogenesis of carcinoma, we do not believe their presence is necessary for the development of hepatocellular carcinoma. (HEPAM M G Y 1992;16:1372-1381.)
confirm malignancy (8).In addition, the Japanese have found MRNs to contain foci of well-differentiated HCC (4,9-11)and have thus come to consider MRNs to be risk factors in the pathogenesis of HCC (1,4, 5, 8, 11). Few investigations, however, have studied the incidence of MRNs and their role in the pathogenesis of HCC in a population without the predominant HBV infection. In this study, we examined 110 sequential liver explants with cirrhoses of various causes to establish the incidence of MRNs and to examine their differentiation from and association with HCC.
MATERIALS AND METHODS We serially sectioned 110 consecutive cirrhotic liver explant specimensat 0.5-cm to 1.0-cmintervals and examined them for the presence of nodules measuring 0.8 to 3.5 cm in greatest diameter (Fig. 1).We used 0.8 cm as the minimum acceptable size, observingthe example of Terada, Hoso and Nakanuma (5) and Terada et al. (9) rather than the l-cm minimum used by Furuya et al. (4) because we found that, on gross examination, nodules of this size stood out significantly against the background liver in micronodular or mixed nodular cirrhoses. In macronodular cirrhoses with very large nodules, this cutoff point may not be valid, and one might only sample nodules of size that had a different color or shape than the other A high incidence of small HCC has been noted in this nodules rather than all nodules. In our cases, this was not an association with cases of HBV infection in Japan (1). issue. Samples from each explant, and every nodule found, Investigators there have used radiographic techniques were fixed in formalin; embedded in paraffin; sectioned at to screen patients with HBV in efforts to identify these 5 km; stained with hematoxylin and eosin, Snook's reticulin small HCCs for early therapy (2). Early therapy reg- and Perl's iron stains; and examined by light microscopy by imens such as hepatectomy, embolization and percuta- L. Ferrell. Each cirrhotic liver was categorized as to origin neous ethanol injection (3) for small lesions have (based on pathological findings, clinical information or both); nodule size (>go% of nodules < 3 mm = micro; 50% of resulted in markedly improved survival rates. nodules > 3 mm = macro; or mixed nodular); iron deposition; In an attempt to make such early diagnoses, Japanese investigators have noted the presence of large regener- and the presence of small-cell or large-cell change of hepato(so-called dysplasia). Small-cell change was defined as ative nodules within cirrhotic livers, so-called macrore- cytes clusters of smaller-than-normal hepatocytes with increased generative nodules (MRNs)or adenomatous hyperplasia nuclearkytoplasmic ratios (Fig. 2A); large-cell change was (1-71, to be a common finding. These benign nodules defined as larger-than-normal hepatocytes with enlarged and cannot be distinguished from malignant nodules by irregular nuclei (Fig. 2B).The extent of small-cell or large-cell radiography, so a histological diagnosis is necessary to change was subjectively graded on a scale of 1 to 3, with 1 = rare, 2 = easily found and 3 = abundant and prominent. The presence of Mallory hyaline and fat was also noted. Each nodule was examined with hematoxylin and eosin h i v e d January 6, 1992; accepted July 27, 1992. stain for the presence of portal tracts or bile ducts; Mallory Address reprint requests to: Linda Ferrell, Department of Pathology, hyaline; small, large, or clear cell change; bile; fat; infiltrative University of California Medical Center, San Francisco, CA 94143-0102. or ill-defined borders; multilobularity; irregular or dilated 31/1/41572 1372
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FIG 1 Explanted liver with mixed nodular cirrhosis and inarrorcgenerati\e nodule The nodule was dark green compared with the more yellow-green background liver
FIG.2. Size differences exist in the hepatocytes in both panels although the photos are the same magnification. (A) Small-cell change (center1 x 325 ). 1 B 1 Large-cell change Note enlarged hepatocytes with large, usually irregularlyshaped nuclei as compared with an adjacent normal-sized hepatocyte ~(irroii'i(1-1 & E. original magnification x. 325).
(H& E, original magnification
sinusoids; and intranodular fibrosis. A nodule was classified as simple MRN if the architectural features were regenerative in nature with a fairly normal reticulin pattern, which, for the most part, was indistinguishable from the background liver (Fig. 3). A nodule was designated at-ypical (borderline HCC or questionable low-grade HCC) if the architectural features were distorted by foci of irregularly thickened cell plates (greater than two cells thick) (Fig. 4) with an associated loss of reticulin. suggesting an abnormal focus ( 4 ) . A thickened cell plate was defined as a small isolated focus of thickening of one or two plates in the same location, suggesting an abnormal prolifer-
ation, but limited in size and orientation as an ill-defined process. A nodule with a definitive trabecular (Fig. 51, pseudoglandular (Figs. 6 and i'),compact or scirrhous pattern was classified as HCC, according to the World Health Organization classification ( 12).Trabeculae were specifically defined in this study as zones of cell plates greater than two cells thick involving several plates in the same area, leading to a pronounced difference in architecture from that of surrounding normal or regenerative plates. Small-cell and large-cell dysplasia-like changes were noted separately and did not influence the classification of nodules (We did not use the
1374
X
FERRELL ET AL.
HEPATOLOGY
FIG.3. Macroregenerativenodule, simple type, with uniform cell plate architecturetypical of a cirrhotic nodule (H& E, original magnification 125).
FIG.4. Atypical macroregenerativenodule with irregular, ill-definedcell plates and small-cell change. The focal granularity of the cells is due to iron (H & E, original magnification x 165).
type I and type I1 designations for MRNs [4J or the HCC
A reticulin stain of each nodule was examined for the absence grading system developed by Japanese investigators [l, 81 of or decrease in staining intensity compared with the because we were not trained in the criteria for such grading surrounding cirrhotic liver (10) (Fig. l l ) , for the presence of and we do not necessarily agree with their nomenclature of thickenedcell plates (greater than two hepatocytes in width) or these lesions.) Infiltrating or irregular edges were defined as any other architectural abnormalities such as the formation of the direct extension of a proliferative focus within the nodule microacini (or pseudoglands) or fragmented trabeculae lined into another portion of the nodule or adjacent tissue (Fig. 8). by endothelial cells (10,13). Iron stain was examined on a scale The intranodular fibrosis consisted of fibrous septa, usually of 1 to 4 + for the quantity of iron deposits in the nodule thin, that separated cell plates, and were not associated with a compared with that present in the background liver. portal tract, bile duct or vessels (10) (Fig. 9), but could result Fisher’s exact test (14) was used to test for significant in an appearance of multilobularity within the nodule (Fig. 10). associations regarding HCV infection, presence of MRNs and
Vol. 16. No. 6. 1992
1375
FIG.5 . (A) HCC with broad trabecular lined by entiothelial cells. small-cell change and wide sinusoidal spaces. ( H & E, original magmfication 325). ( B ) Reticulin stain of same lesion shows t h r ahsence ( i f lincvw itaining at the edges of trabeculae. typical of HCC (reticulin, original magnification 325 I . x
presence of HCC. One-sided p values were used because of the ambiguity in defining the second tail for this test ( 151. Because of the small numbers Of atuYPicalMRNs and "CCS, an exact method (16)was also used to test for trends i n the proportions of nodules exhibiting various histological features. HCV infections were confirmed by the polymerase chain reaction previously described I 171. Patients with alcoholic and cryptogenic cirrhosis were confirmed to he negative for HCV by the polymerase chain reaction. All patients had a minimum of 5 mo clinical follow-up
RESULTS The orisns for the cirrhoses, the size of the and the extent of smdl-cell Or lmge-ceu change found in each explant are listed in Tables 1-3. Forty nodules rangmg in diameters from 0.8 to 3.5 cm from l 9 patients (17.3% of 110 patients) were found (Fig. 1 ) . The number of nodules Per Patient, their dlstribution and type (simple, atypical or HCC) and hackground liver findings in each patient are listed in
1376
FERRELLETAL
HEPATOLOGY
FIG.7. Formation of pseudoglands, or microacini (urrowsl, with small-cell change in a liver 3 wk after transplantation. Most of the other hepatocytes are of normal size (H & E, original magnification x 325).
FIG.8. Infiltrative edge of HCC (center),with well-differentiated HCC on right and adjacent nodule on left. Note the small clusters of atypical cells as seen in the HCC infiltrating the fibrous stroma outside of the main nodule mass (H & E, original magnification x 63).
Table 4. Twenty-five nodules found in 12 patients were classified as simple, 6 nodules in 3 patients were classified as atypical and 9 nodules in 5 patients were classified as HCCs only. In addition, three nodules contained both HCC and a residual rim of tissue with architectural and cytological features of MRN (one simple, two atypical) for a total of 12 HCCs in eight patients. Four patients had both MRN and HCC, and patients with at least one MRN had a much higher rate of HCC (4 of 15, 26.3%) than those with no MRNs
(4 of 95, 4.2%,p = 0.012). One of the three patients with atypical nodules and four of the eight patients with HCC had cirrhosis caused by HCV. Thus having HCV as the cause of the liver disease was associated with an increased chance of having a nodule of any type (30%vs. 12.5%for all others, p = 0.034) and of having HCC (13% vs. 5%, p = 0.14). The incidence of HCC diagnosed as this size of nodule was 12 of 43 (28%), with the end result that 8 of 19 patients (42%) with nodules (7% of the 110 total cases reviewed) received
Vol. 16, No. 6, 1992
MA(’KORE(;ENEK;\TIVE NOIIL-LES VS I ICC
1377
FIG. 9. Intranodular fibrosis of H(’( Thr thln fibrous septa separate trabeculae of the tumor and do not contmn bile ducts or other portal structures This HCC also shows fattc c hmge rH & E: oriplnal magnlficatlon 6 3 1
a malignant diagnosis. Eleven of the 19 patients (58%; 10% of the total cases reviewed) received a benign diagnosis of MRN only. Pathological features of the MRNs and HCCs are listed in Table 5 . Portal tracts or isolated bile ducts were not present in HCCs. Mallory hyaline, clear cell change and bile were seen in both types of MRNs and in malignant lesions. All six atypical nodules had small-cell change, five had foci of decreased reticulin and four had foci of thickened cell plates, but all three features were
never seen in the same focus in a given nodule. The lack of or marked decrease in reticulin (Figs. 5 and 111, lack of iron and trabeculae formation were the only features seen in all of the HCCs; however, many of the benign nodules lacked iron also. We considered the definitive formation of trabeculae in the nodule as the best diagnostic criterion (or definition) for carcinoma. In doing so, we noted an attendant loss of reticulin framework in the malignant nodules (p < 0.001). An infiltrative border or multilobularity and intranodular
1378
FERRELL ET AL.
HEPATOLOGY
FIG.11. Core biopsy specimen of cirrhotic liver with well-differentiated HCC, reticulin stain, diagnosis confirmed on explant tissue. (A) Reticulin of the background liver is well demonstrated and distinct, and the cell plates of the cirrhotic nodule do not fall apart (reticulin, original magnification x 125). (B) In contrast, the HCC has essentially no reticulin framework, and consequently breaks into smaller, dissociated cell plates, many only two to three cells in thickness, in this field. Other fields had foci of fragmented trabecular plates up to five cells thick. Note the small-cell change (reticulin, original magnification x 1251.
TABLE 1. Origin of cirrhotic explants AU livers (n =
Livers with nodules (n = 19P
110)
Origin
Number
Percentage
Hepatitis C virus Alcohol Cryptogenic Autoimmune Sclerosing cholangitis Biliary atresia HBV PBC Hemochromatosis
30 25 19 7 10 5
27 23 17 6 9 4
5
4
7 2
6 2
Number
Percentage
~
47 (30% of hepatitis C virus) 11 (8% of alcohol) 26 (26% of cryptogenic) 5 (14% of autoimmune)
5 (14% of PBC) 5 (50% of hemochromatosis)
"Nodules defined as measuring 0.8 to 3.5 cm.
TABLE 2. Type of background cirrhosis AU livers (n =
110) Livers with nodules (n
Description Number Percentage Number ~~
~~
Macronodular Micronodular Mixednodular
4 28
78
=
19)"
Percentage
~~
4 25 71
2
3 14
10 (50% of macro) 16 (11% of micro) 74 (18% of mixed)
"Nodules defined as measuring 0.8 to 3.5 cm.
unremarkable MRNs (Fig. 2A). Small-cell change was also prevalent in the areas of trabeculae or pseudoglands in the HCCs (Fig. 6A). So-called large-cell dysplasia was a common finding (Fig. 2B).In our study, 65 (59%)of the examined livers, including 14 (74%)with nodules (MRNs and HCCs) and 5 (62%)with HCC, had some degree of large cell change. Twenty-six (60%) of the nodules showed large-cell change, including 4 of 6 atypical MRNs (67%) with large-cell change and 8 of 12 (67%) of the HCCs.
DISCUSSION fibrosis were also significantly more typical of HCC In our study, the incidence of patients who had (p < 0.001). We were able to identify clusters of small-cell change nodules that ranged in size from 0.8 to 3.5 cm in in 52% (13 of 25) of simple MRNs, 100% (6 of 6) of diameter was 17.3%; 7% (eight patients) had diagnoses atypical MRNs and 75% (9 of 12) of the HCCs. Single or of HCC and the remaining 10% proved to be MRNs. Two multiple clusters could be present in otherwise totally studies from Japan showed an incidence of MRNs of
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MA('H0KEGENERA'rIVE NODULES VS HCC
Vol. 16, No. 6, 1992
TABLE 3. Cytological changes in background cirrhosis All livers
CyLOlogical atypia
1.
Small-cell change Large-cell change
-13
7
22
:34
2-
Livers with nodulesf
3 .
Total (percentage of 110 livers)
1+
24
3 4-
0 9
14 11:?%I
4
2
0
65 (60%I
2
8
4
Total (percentage of 18)
6 (32%) 14 ( 73%)'
1 + = rare, 2 I- = moderate number5 .1 ahundant "Nodules defined as measuring 0 8 ti) 3 5 cm *Expressed as number of livers 'Five of eight livers w t h HCC included 4
TABLE 4. Type of nodules and relationship to origin and type of cirrhosis Number of patients
Number of noduledpatient
Morphological type
Origin of cirrhosis
Size of cirrhotic nodules
4
1
S
3 mixed, 1 micro
3 HCV, CC
3 3 1 1 1 1 1
)
S
i
HC"
ti 1
S
2 mixed, micro Mixed Macro Mixed Macro Mixed Mixed Mixed Mixed Mixed Mixed
HCV, ETOH, PBC CC, HCV, ETOH Autoimmune H CV C3C HCV HCV
1
1 1 1
s S
:i 1
> > I 1
2A. I-ICt' in A s. 4 €lC(k A. k1CC i n A 2 II(Ts HCC' in S A
c:c
HCV
(XI Hema
S = simple macroregenerative nodule; A at-ypical macroregenerative nodule; Macro = macronodular; Mixed Micro = micronodular; CC = c~yptogeniccirrhosis: ETOH alcohol: Herno = hemochromatosis.
14.2% (49 of 345 cases [41, with nodules measuring greater than 1 cm), and 37% (53 of 141 cases [61, with nodules measuring greater than 0.5 cm); thus the incidence of these lesions in our study was comparable to those seen in Japan despite the fact that our population had a different etiological pattern. Most patients in our study had HCV (27%) or alcoholic (23% or cryptogenic types of cirrhosis ( 16%1. The patients in the Japanese studies. on the other hand, showed a predominance of HBV. In Furuya et al.'s study (4), 38.8% of the patients with livers examined for MRNs ( a total of 345) were positive for HBsAg and 32 of the 49 MRNs (65%) found were in livers that also contained foci of HCC. The exact number of HBV-positive patients with both an MRN and a focus of HCC was not stated, but the authors did state that 49% of patients with an MRN (with or without associated HCC) were HBV positive. In contrast, in our study only 6 of 43 (14%) of the MRNs were found in livers with foci of HCC. Only 4% (five patients) were HBV positive, and none of our HBV patients had an MRN or a focus of HCC. This difference may suggest that the comparatively lower incidence of HCC associated with the MRNs in our study may in part be caused by the absence of HBV (18-21) in these livers. Additional studies with more cases may be necessary to confirm this relationship. Another possible explanation could be the use of dif-
=
mixed nodular;
ferent criteria for the diagnosis of carcinoma in Japan than in our study, which may result in a lower incidence of malignancy. It is interesting to note that four of the eight patients with HCC in our study were HCV positive, and two of these patients had MRNs. In addition, 9 of the 19 patients with nodules were HCV positive (47%),whereas only 27% of the total number of patients were HCV positive. Alcohol, on the other hand, was the cause for cirrhosis in 23% of the patients, but only two of these This prevalence of MRN patients had a nodule (11%). and HCC in the HCV group may suggest a similar association of HCV with these lesions as seen in the HBV population. Evidence suggesting that HCV may play a causal role in the development of HCC has been reported (22). Various histopathological features of MRNs and small HCCs were examined in the Japanese studies, and it was shown that HCCs have an increased incidence compared with simple MRNs if so-called small-cell change (4, 8, 23), lack of iron deposits (9, 111, trabeculae (1, 101, pseudoglands or microacini (1, 8, 101, peritrabecular fibrosis (10) and loss of reticulin framework occurred ( 10). MRNs and HCCs may both show clusters of clear cells that contain Mallory bodies (5,24-26) and increased amounts of bile (10). Our study supports some but not all of these findings (Table 5). Our main difficulty was in distinguishing small-cell
1380
FERRELL ET AL.
TABLE 5. Pathological features of MRNs and small HCCs
Portal tractsbile ducts Lack of ironb Mallory hyaline Small-cell change Large-cell change Increased bile Clear-cell change, focal Clear-cell change, diffuse Fatty change Irregular sinusoids or peliosis Decreased reticulin' Infiltrating borders or multilobular areas of atypia' Intranodular fibrosisd Plates more than two cells thick
HCC
Simple (n = 26)
Atypical (n = 6)
14 15 14 13 14 9
0 12 4 9
5
3 4 1 6 4 1 4
3 7
0.002 0.019 0.170 0.150 0.600 0.470 0.019
0
0
2
0.073
4
0 2
6 2
0.043 1.00
5
11 9
< 0.001
6 12
< 0.001 < 0.001
4 3 0
0 0
1
1 4
(n = 12)
8
pValue"
< 0.001
MRN = macroregenerative nodule; Simple = nodules without architectural abnormalities, resembling routine regenerative nodules; Atypical = nodules with focal architectural abnormalities including broader cell plates consistent with a proliferative focus within the nodule. "Test for either increasing or decreasing proportions, with atypical considered to be midway between simple and HCC. The p values for the comparison of atypical vs. HCC for the last three features are 0.032, 0.20 and 0.10, respectively. *No iron or decreased over the background cirrhotic liver. 'Edge of nodule shows focal irregularity, extending beyond the confines of the border of the lesion in an infiltrative manner or has the appearance of a multilobular lesion. dFibrosis not associated with a portal tract or bile duct, consisting of fibrous septa separating cell plates.
dysplasia from regenerative changes with small hepatocytes or compression of hepatocytes by regenerative foci
(Figs. 2, 5). At times, small cells would appear to be compressed by adjacent regenerating tissue and at other times would seem to be admixed with a nodular regenerative focus. We also found a rare pseudogland or microacini in these foci. We recognize that Kondo et al. (8) have proposed that lesions with small-cell dysplasia and microacini should be classified as foci of HCC even in the presence of normal cell plates, but we do not necessarily agree that this should be the minimal malignant criterion. We have seen numerous examples in transplanted livers of regenerative foci with small cells and microacini that were histologically similar to the described foci of small-cell dysplasia (Fig. 7). These findings are especially prominent the first few weeks &er transplant, when the liver is actively regenerating to replace cells lost during transport and perfusion. We suggest that many of these small foci in MRNs may represent similar regenerative processes and not necessarily a malignant process. On the other hand, we agree that larger zones of small-cell change should be ex-
HEPATOLOGY
amined closely for the underlying architectural abnormalities associated with the trabecular, pseudoglandular or compact types of HCC because small-cell change can be a typical feature of HCC (Fig. 5 ) . Large-cell change (Fig. 2B) was also not a discriminatory finding because it was seen abundantly in the MRNs and in the cirrhotic livers with and without nodules. Thus the presence of large-cell change as an isolated finding is not a diagnostic marker for distinguishing cirrhosis from MRN or HCC. In addition, our findings do not confirm a definitive preneoplastic role for large-cell dysplasia, and one might suggest that at least some of the large atypical cells represent a form of regenerative or even degenerative change. Of the remaining histological features of MRNs and HCCs (Table 51, the loss or absence of reticulin, thickened cell plates (more than two cells), intranodular fibrous septa and infiltrative borders or multilobular areas of atypia may be the features that would most raise the suspicion for HCC because of the high prevalence of these findings in the HCCs but not in the simple type of MRNs. Whether an atypical lesion that shows some of these features should be considered low-grade HCC is debatable. Standardization of criteria for the classification of such atypical nodules would help in this regard. The presence of multiple large nodules was also a confusing factor. A multifocal process could represent multifocal HCC, as was the case in two patients. Three of our patients, however, had one or more nodules of HCC in addition to one or more MRNs, and six patients had more than one MRN. Because lesions of this size and/or multiplicity could potentially be discovered with radiographic techniques (2), pathologists should be aware of the relatively high frequency of benign nodules and be prepared to differentiate MRNs from HCCs. In addition, in these instances of multifocality, one might be faced with the dilemma of which nodule to sample or remove because the pathological finding of any one nodule may not be representative of the pathological finding of the other nodules. A problem that will most likely arise in the diagnosis of such nodules will be the size and type of sample obtained. Because of the relatively small size of the nodules, many biopsy specimens are likely to contain surrounding cirrhotic tissue. Fine-needle aspiration biopsy (27,28)with smear examination alone may not be sufficient to make the diagnosis because the presence of large-cell or small-cell dysplasia within an MRN or the surrounding liver parenchyma could be misinterpreted as HCC, especially on less cellular specimens. A cell button of a fine-needleaspiration biopsy sample or a core biopsy sample allows for better examination of the architectural features (e.g., trabeculae formation and loss of reticulin) (Fig. 11) that can distinguish benign lesions from well-differentiated HCCs. A core biopsy specimen might provide a better sample than a cell button, but, as with a fine-needle aspiration biopsy sample, it is imperative to obtain representative material to avoid sampling error. Thus the pathologist will
MA(’K( )KEGENERATIVE NODVLES VS HC‘C’
Vol 16. No 6, 1992
no doubt need to communicate with the gastroenterologist and radiologist to ensure adequate sampling of the region in question. In summary, we conclude that MRNs are relatively common findings outside of Japan and that these benign lesions may be more easily separated histologically from well-differentiated HCC by their architectural features in conjunction with the cytological features rather than by cytology alone. We support the issue that the MRNs may themselves predispose to the formation of HCC, but they do not represent the only predisposing factor because our study contained several small HCCs without associated MRNs. We also suggest that cofactors such as HCV may enhance the risk for HCC with or without the presence of an underlying MRN.
ri
9
10
11
12
Acknowledgments: We thank David Geller for editorial advice and assistance and Peter Bacchitti (from the Liver Center, University of California. San Fran- 1 1 cisco, supported by Liver Core Center Grant AM-26743 1 for the statistical analysis. We also wish to acknowledge 14 the International Liver Pathology Study Group, consisting of the following members: C. Balabaud, Bordeaux 15 Cedex, France; K. Bardadin, Warsaw. Poland; P. 16 Bioulac-Sage, Bordeaux Cedex, France; J. Crawford, Boston, MA; R. Colombari, Verona, Italy: P. Dhillon, London, UK; L. Ferrell, San Francisco. CA; Y. Na- 17 kanuma, Kanazawa, Japan; B. Portmann. London. UK; J. Rode, Melbourne, Australia; P. Scheuer. London, LJK; D. Snover, Minneapolis, MN; N . Theise, New York, NY; 18 S. Thung, New York, NY; W. Tsui, Kowloon, Hong Kong; and D. VanLeeuwen. Amsterdam, The Nether- 19 lands. This group discussed the issue of MRNs at their meeting in London in July 1990 (generously supported 20 by Ortho Diagnostic, Dako and Roche Products) and provided valuable input and inspiration for this study. REFERENCES 1. Sakamoto M, Hirohashi S. Shimosato Y. Early stages of’multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol 1991:22:172-178. 2. Rapaccini G , Pornpili M. Caturelli E.Anti M. Aliottu A, Cedrone A. Amadei E, 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. 3. Yamasaki S, Hasegawa H, Makuuchi M , l’adatoshi ‘I‘. Kosuge ‘1’. Shimada K. Choice of treatments for sinall hepatocellular carcinoma: hepatectomy, embolization. or ethanol injection. J Gastroenterol Hepatol 1991:6:408-413 4. Furuya K, Nakamurd M. Yamamoto Y. ‘I’ogei K, Otsuka H. Macroregenerative nodule of the liver. a clinicopat hologic study of 345 autopsy cases of chronic liver disease. Cancer 1988;61:99-105. 5. Terada T, Hoso M, Nakanuma Y. Mallor?. body clustering in adenomatous hyperplasia in human cirrhotic livers: report of four cases. Hum Pathol 1989;20:886-89(1. 6. Wada K, Kondo F, Kondo Y. Large regenerative nodules and dysplastic nodules in cirrhotic livers. a histopathologic study. HEPATOLKY1988;8:1684- 1688. 7 . Kondo F, EbaraM, Sugiura N, Wada K. f i t a K. Hirooka N, Nagoto Y, et al. Histological features and clinical course of large regener-
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