Hepatocellular carcinoma associated with focal nodular hyperplasia-Report of a case with clonal analysis

Abstract We describe a hepatocellular carcinoma

par-tially surrounded by focal nodular hyperplasia in a

65-year-old female patient. In order to clarify the

relation-ship of the hepatocellular carcinoma and the adjacent

fo-cal nodular hyperplasia, clonal analysis was conducted.

The clonal analysis was based on the methylation pattern

of the polymorphic X-chromosome-linked androgen

re-ceptor gene (HUMARA). The allelic bands from the

am-plification of the focal nodular hyperplasia and of the

he-patocellular carcinoma showed a significant reduction in

the intensity of one of the two alleles as compared with

two alleles of equal intensity in the buff coat after HhaI

digestion, which indicated that these two parts were

monoclonal. However, the inactivated allele in the focal

nodular hyperplasia and that in the hepatocellular

carci-noma were not identical. Therefore, the focal nodular

hy-perplasia and hepatocellular carcinoma probably derived

from the clonal expansion of two different clones.

Keywords Focal nodular hyperplasia · Hepatocellular

carcinoma · Androgen receptor gene · HUMARA ·

Clonality

Introduction

Focal nodular hyperplasia (FNH) is a common benign

hepatic tumor. It is generally considered to be a

hyper-plastic response to an abnormal blood supply [13].

How-ever, its nature and pathogenesis are still controversial. It

has been recently shown to be a clonal proliferative

dis-ease using HUMARA (methylation pattern of the

poly-morphic X-chromosome-linked androgen receptor gene)

analysis [2]. Its associations with fibrolamellar

hepato-cellular carcinoma (HCC) were also reported [1, 11, 12].

Although some reports indicated an association between

FNH and HCC, most authors did not consider a

pathoge-netic correlation between them. In this report, we

de-scribe a FNH arising at the periphery of a HCC. Both

le-sions arose from clonal expansion of two different

clones.

Clinical history

A 65-year-old Taiwanese female patient, with a history of perni-cious anemia for several years, was noted to have hepatomegaly in a routine follow-up. Laboratory data were as follows: aspartate aminotransferase (AST) 240 U/l (normal <34 IU/l), alanine ami-notransferase (ALT) 100 U/l (normal <36 IU/l), alkaline phospha-tase 151 U/l (normal <96 IU/l), γ-glutamyl transferase 398 U/l (normal <96 IU/l), hemoglobin 9.4 g/dl, and white blood cell count 6800/cc. Serum α-fetoprotein was 2384 ng/ml (normal <5 ng/ml). Serum hepatitis B virus surface antigen and anti-hepa-titis C virus antibody were negative. Image studies, including computed tomographic scan, abdominal ultrasound, and angiogra-phy, showed a huge hyperechoic, heterogeneous, hypervascular liver tumor occupying the right lobe and medial portion of the left lobe with central necrosis. The size of the spleen was within nor-mal limits. There were no tumor thrombi within the portal veins. After the liver tumor was resected, the level of serum α -fetopro-tein returned to the normal range.

Materials and methods

Representative sections were taken from the surgical specimen and fixed in formalin and embedded in paraffin. Histologic sec-T.-C. Chen (

✉

Department of Pathology, Chang Gung Memorial Hospital, 5 Fu Shin Street, Kwei San, Tao Yuan, Taiwan

e-mail: [email protected]

Tel.: +886-3-3281200 x2742, Fax: +886-3-3280147 T.-B. Chou

Department of Zoology, National Taiwan University, Taipei, Taiwan

L.-L. Hsieh

Department of Public Health, Chang Gung University, Tao Yuan, Taiwan

Y.-H. Wu Chou

Human Molecular Genetics Laboratory,

Chang Gung Memorial Hospital, Tao Yuan, Taiwan Virchows Arch (2001) 438:408–411

DOI 10.1007/s004280000348

C A S E R E P O R T

Tse-Ching Chen · Tze-Bin Chou · Kwai-Fong Ng

Ling-Ling Hsieh · Yah-Huei Wu Chou

Hepatocellular carcinoma associated with focal nodular hyperplasia

Report of a case with clonal analysis

Received: 31 May 2000 / Accepted: 18 September 2000 / Published online: 19 December 2000 © Springer-Verlag 2000

409

Fig. 1 The liver tumor was grossly composed of two different portions. The lower part was yellow and nodular, and the upper portion was brown with a large area of necrosis

Fig. 2 The yellow portion of the liver tumor was composed of large hyperplastic hepatocytes with short fibrous septa containing malformed vessels and bile ductules. ×200

Fig. 3 The brown portion of the liver tumor showed a classical hepatocellular carcinoma arranged in trabecular and acinar pat-terns. ×100

tions were stained with hematoxylin and eosin, periodic acid-Schiff with diastase digestion, Masson trichrome, reticulin, Perls’iron, and Victoria blue stains.

Polymerase chain reaction and clonal analysis

The liver tumor was grossly composed of two different parts, i.e., yellow and brown parts. Fresh tissue samples were dissected from the yellow and brown parts of the liver tumor. Peripheral blood of this patient was also available, and the buffy coat was collected for analysis. High molecular weight DNA was extracted and re-suspended in 20 µl of water and split into two parts. One part was digested with 20 U HhaI restriction enzyme in a 40-µl reaction mixture at 37°C for 24 h.

Clonal analysis in the present study is based on HUMARA. The primer sequences used for the amplification of the HUMARA gene were 5'-TCCAGAATCTGTTCCAGAGC-3' and 5'-TGGGG-AGAACCATCCTCACC -3', as previously described [6]. Each DNA sample (2 µl) was added to a 50-µl reaction mixture contain-ing 100 ng of each primer, 200 µM dNTPs, and 0.3 U Taq poly-merase in a standard polypoly-merase chain reaction (PCR) buffer. One

of the primers was end-labeled with [γ-32_{P] dATP. Thirty cycles of} amplification were carried out using cycling parameters of 95°C for 30 s, 64°C for 30 s, and 72°C for 30 s. Each PCR product (2 µl) was added to an equal volume of formamide containing 0.1% bromophenol blue and 0.1% xylene cyanol, loaded onto a 7 M urea–polyacrylamide gel, and electrophoresed at 60 W for 4 h. The gel was dried and autoradiographed at –70°C.

Quantitative measurement of the PCR product bands was also performed by using a densitometer, as described by Paradis et al. [10]. In brief, the peak intensities of the two alleles (alleles 1 and 2) were measured for each specimen. A corrected ratio (CR) was first assessed by dividing the ratio (allele 1/allele 2) of the digest-ed samples by HhaI by the ratio (allele 1/allele 2) of the non-di-gested sample. The use of CR corrects for the preferential amplifi-cation of one allele that might occur if the alleles differ markedly in length. A final clonality ratio was determined by dividing the CR of the lesional DNA by the CR of the non-lesional DNA. The ratio was inverted if necessary to obtain a value up to one. Ac-cording to Paradis et al. [10], a final ratio of 1.5 corresponded to the presence of 25% clonal DNA in a polyclonal background. This value (1.5) was chosen as the threshold of sensitivity of a signifi-cant number of clonal cells.

Results

Pathologic findings

The resected liver tissue measured 20

×

18

×

12 cm and

weighed 2900 g. There was a well-circumscribed but not

encapsulated tumor mass (Fig. 1) measuring 20

×

14

×

11 cm. It was grossly composed of two different parts.

The larger part of the liver tumor was yellow with a

nod-ular appearance. The other part was brown and soft with

a large area of necrosis. An aberrant vessel penetrated

through the yellow portion to the brown part. However,

no central scar was found in the yellow part. The

non-tu-mor part had no obvious nodularity.

Microscopically, the yellow portion was composed of

large hyperplastic hepatocytes with mild anisonucleosis.

There were scattered fibrous septa and abnormal portal

tracts with bile ductular proliferation and thick-walled

ar-terioles (Fig. 2). Mallory bodies and fatty change were

present. Reticulin stain showed that the liver cell plates

were two to three cells thick. Focal Kupffer cell siderosis

was identified with Perls' iron stain. The brown portion

(Fig. 3) showed a classical HCC arranged in trabecular

and acinar patterns with a large area of necrosis. No

fi-brous septum was present between the two portions of the

liver tumor. The non-tumor part showed some fibrous

sep-ta but no cirrhotic change. There were grade III Kupffer

cell siderosis and young hematopoietic cells in sinusoids.

The result of the clonal analysis is shown in Fig. 4.

Without restriction enzyme digestion by HhaI, two

allel-ic bands with equal intensity were observed in this

pa-tient, which indicated that the HUMARA gene of this

patient was heterozygous and can be analyzed. After

HhaI digestion, the allelic bands from the FNH and the

HCC showed a significant reduction in the intensity of

one of the two alleles compared with two allelic bands

with equal intensity in the buffy coat. Thus, the HCC and

FNH were interpreted as being monoclonal. However,

the inactivated allele in the FNH and the one in the HCC

were not identical. Therefore, the clonal origins of the

FNH and HCC were different.

The final ratios of FNH and HCC using densitometric

assessment of the PCR product bands were greater than

ten, indicating the presence of 25% or more of clonal

DNA. Thus, the quantitative analysis further confirmed

the clonality of the FNH and HCC.

Discussion

The liver tumor described in this report was grossly

composed of two different parts with different histologic

features. The yellow part was composed of large

hyper-plastic hepatocytes with nodular abnormal architecture,

malformed vessels, and bile ductular proliferation.

Al-though the central scar was absent in this portion, the

le-sion fulfilled the morphologic diagnostic criteria of a

FNH [8]. The large cell change in this lesion could

re-present an adaptive reaction to prolong cholestasis [7].

The brown portion was a classical HCC. The

associa-tions of HCC with adjacent FNH were rarely described

[1, 11, 12]. In contrast to these reported cases, the HCC

component in our case was not a fibrolamellar variant

but a classical type. The FNH could have developed in

our case secondary to the feeding artery of the HCC.

FNH has been demonstrated to be a clonal

proliferat-ing disease by HUMARA analysis [2]. Clonality can be

assessed using the HUMARA assay when monoclonal

cells comprise more than 25% of the total cell population

in the specimen analyzed [10]. The results in the present

study also showed that the FNH (yellow part) was

mono-clonal, as evidenced by a significant reduction in the

in-tensity of one allele as compared with the two alleles of

equal intensity in the buffy coat. However, a clonal

dis-ease is not invariably equivalent to a neoplastic process

[3, 4]. There are two major mechanisms that could

ex-plain the occurrence of a uniform pattern of X

HhaI digestion, two allelic bands with equal intensity were

recog-nized in the three specimens. In the presence of HhaI digestion, the allelic bands from the focal nodular hyperplasia and the hepa-tocellular carcinoma showed a significant reduction in the intensi-ty of one allele as compared with two alleles of equal intensiintensi-ty in the buffy coat (C), indicating monoclonal origins of these two le-sions. However, the inactivated allele in the focal nodular hyper-plasia and that in the hepatocellular carcinoma were not identical

3. Iannaccone P M, Weinberg W C, Berkwits L (1987) A proba-bilistic model of mosaicism based on the histologic analysis of chimaeric rat liver. Development 99:187–196

4. Kawai S, Imazeki F, Yokosuka O, Ohto M, Shiina S, Kato N, Omata M (1995) Clonality in hepatocellular carcinoma: analy-sis of methylation pattern of polymorphic X-chromosome-linked phosphoglycerate kinase gene in females. Hepatology 22:112–117

5. Kopp P, Kimura ET, Aeschimann S, Oestreicher M, Tobler A, Fey MF, Studer H (1994) Polyclonal and monoclonal thyroid nodules coexist within human multinodular goiters. J Clin En-docrinol Metab 79:134–139

6. Mashal R D, Lester S C, Sklar J (1993) Clonal analysis by study of X chromosome inactivation in formalin-fixed paraf-fin-embedded tissue. Cancer Res 53:4676–4679

7. Natarajan S, Theise ND, Thung SN, Antonio L, Paronetto F, Hytiroglou P (1997) Large cell change of hepatocytes in cir-rhosis may represent a reaction to prolonged cholestasis. Am J Surg Pathol 21:312–318

8. Nguyen BN, Flejou JF, Terris B, Belghiti J, Degott C (1999) Focal nodular hyperplasia of the live: a comprehensive patho-logic study of 305 lesion and recognition of new histopatho-logic form. Am J Surg Pathol 23:1441–1454

9. Paradis V, Laurent A, Flejou JF, Vidaud M, Bedossa P (1997) Evidence for the polyclonal nature of focal nodular hyperpla-sia of the liver by the study of X-chromosome inactivation. Hepatology 26:891–895

10. Paradis V, Laurendeau I, Vieillefond A, Blanchet P, Eschwege P, Benoit G, Vidaud M, Jardin A, Bedossa P (1998) Clonal analysis of renal sporadic angiomyolipomas. Hum Pathol 29:1063–1067

11. Saul SH, Titelbaum DS, Gansler TS, Varello M, Burke DR, Atkinson BF, Rosato EF (1987) The fibrolamellar variant of hepatocellular carcinoma: its association with FNH. Cancer 60:3047–3055

12. Saxena R, Humphreys S. Williams R, Portmann B (1994) Nodular hyperplasia surrounding fibrolamellar carcinoma: a zone of arterialized liver parenchyma. Histopathology 25:275– 278

13. Wanless IR, Mawds C, Adams R (1985) On the pathogenesis of focal nodular hyperplasia of the liver. Hepathology 5:1194– 1200

411

some inactivation in a non-neoplastic lesion. The first

one is the patch concept [3, 4]. If, after cell division,

progeny cells remain adjacent to each other, large

patch-es of cells are formed, all of which contain an identical

pattern of X chromosome inactivation. Lesions arising

from two, three, or more cells within a patch will show

the same pattern of X chromosome inactivation and

ap-pear clonal. Another explanation for the apap-pearance of

clonality is the selection hypothesis [3]. If one cell type

within a multicellular lesion has a growth advantage,

se-lective overgrowth of this cell type over time would

re-sult in a proliferation with an overall monoclonal pattern.

Such a situation has been postulated for multinodular

goiters of the thyroid [5]. Our results support that FNH is

a clonal lesion. However, conflicting results were

report-ed by Paradis et al. [9] using the same method.

There-fore, the nature of FNH is still not settled.

In the present study, our results also showed that the

inactivated allele in the FNH and the one in the HCC

were different. These results indicated that the HCC and

adjacent FNH probably developed through clonal

expan-sion of different clones. Therefore, our study did not

support that the HCC was the product of malignant

transformation from the FNH.

Acknowledgements This work was supported in part by grants NSC 89–2314-B182A-011 and NSC 85–2331-B-182A-012 from the National Science Council, R.O.C. and by the Chang Gung Medical Research Grant CMRP799.

References

1. Davidson BR, Vaisamidakis N, Scheuer P, Hobbs K (1990) Fi-brolamellar carcinoma arising in an abnormal liver (letter). J Hepatol 11:388

2. Gaffey MJ, Iezzoni JC, Weiss LM (1996) Clonal analysis of focal nodular hyperplasia of the liver. Am J Pathol 148:1089– 1096