Alimentary tract and pancreas

Alimentarni trakt i pankreas

1Thanaa El-Sayed Helal,

2Nehal Mohamed El-Koufy,

2Mohamed Khalil Abdel Khalek,

3Manal Hassan Moussa,

4Hesham Abdel Kader Hassan.

1Department of Pathology, Faculty of Medicine,

Ain Shams University, Cairo,

2Department of Padiatrics, Faculty of Medicine,

Cairo University, Cairo,

3Department of Histology, Ain Shams University,

Cairo,

4Departent of Paediatric Gastroenterology and

Hepatology, State University of New York, Health

Science Center, Syracuse , NY,USA.

.

ARCH GASTROENTEROHEPATOL 2003; 22 (No 1 - 2): 12 – 17

Morphometric and DNA

analysis in Egyptian children

with chronic liver

diseases

Morfometrijska i DNA analiza Egipatske dece sa

hroni~nim bolestima jetre

( accepted April 24th, 2003 )

Key Words:

chronic liver disease, morphometry, DNA

analysis, dysplasia.

Abstract

The aim of the present study was to assess the value of morphometric and DNA image analysis in the

assessment of chronic liver diseases in children. Seventy liver biopsies from children with chronic liver

diseases were included in the study (30 infantile cholestasis, 24 chronic hepatitis, 16 glycogen storage

disease, GSD). The biopsies were examined histologically for estimation of the grade of the disease

activity, stage of fibrosis, and degree of dysplasia. DNA ploidy was determined by using Feulgen,s

stained sections and CAS 200 image analyser. Morphometric analysis of the nuclear area, nucleo-cytoplasmatic

(N/C) ratio and the degree of fibrosis (fibrotic index) were also estimated. Although the grade

of disease activity was not significantly diferent among the 3 disease group, the stage of fibrosis was significantlly

more in the cholestatic and hepatitis group than GSD. DNA aneuploidy was observed in 8

out of 70 liver biopsies, 4 with infantile cholestasis, and 4 with chronic hepatitis. The 3 disease groups

were not statistically different regarding the nuclear area or the fibrotic index. However, the N/C ratio

was significantly higher in the cholestatic and hepatitis group than in GSD. Moreover, the semiquantitative

staging of fibrosis was significantly correlated with the qantitative measurement of the fibrotic

index. These results suggest that image analysis especially morphometry is a helpful objective method

in the investigation of chronic liver diseases in children. Liver cell dysplasia, DNA aneupoloidya, and

icreased N/C ratio can be used as risk factor for developing hepatocellular carcinoma in children with

chronic liver diseases either early or later in the life. Infantile cholestasis seems to have premalignant

potential in Egyptian children similar to well known role of chronic viral hepatitis.

Sa`etak

Cilj ove studije je bio da se proceni vrednost morfometrijske i DNA analize u proceni hroni~nih bolesti

jetre u dece. Sedamdeset biopsija jetre dece sa hroni~nim bolestima jetre je uklju~eno u ovo ispitivanje (30

slu~ajeva infantilne holestaze, 24 hroni~nog hepatitisa, l6 glikogenoza, GSD). Biopsije su pregledane histolo{

ki i izvr{ena je procena stepena aktivnosti bolesti, stadijum fibroze, i stepen displazije. DNA-ploidija

je odredjivana kori{}enjem Feulgen-ovim rastvorom bojenih sekcija tkiva jetre i CAS 200 analizatorom

slike. Morfometrijska analiza jedra, nukleo-citoplazmatskog (N/C) odnosa i stupnja fibroze (indeks

fibroze) je takodje vr~ena. Premda stadijum aktivnosti bolesti nije bio zna~ajno razli~it izmedju ispitivane

tri grupe bolesti, stupanj fibroze (fibroti~ki indeks) je bio izra`eniji u grupi holestaznih bolesti jetre i hepatitisima

nego u GSD. DNA aneuploidija je otkrivena u 8 od 70 biopsija, i to u 4 sa infantilnom holestazom

i 4 sa hroni~nim hepatitisom. U sve tri grupe bolesti jetre nije bilo razlika u pogledu procene jedra i

fibroti~kog indeksa. Medjutim N/C odnos je bio zna~ajno ve}i u dece biopsijskim uzorcima dece sa

holestazom i hepatitisom mego u onih sa GSD. Semikvantitativno merenje stadijuma odmaklosti firboze

je bilo u signifikatnoj korelaciji sa kvantitaivnim merenjem fibroti~kog indeksa. Ovi rezultati ukazuju da

posebno morfometrijska analiza uzoraka tkiva jetre je dobra pomo} u ispitivanju dece sa hroni~nim bolestima

jetre. ]elijska displazija, DNA aneuplodija, i povi{en N/C odnos je faktori rizika za nastanak hepatocvelularnog

karcinoma jetre u dece sa hroni~nim bolstima jetre. Infantilna holestaza je ~ini se grupa

bolesti sa najvi{im premalignim popterncijalom u Egipatske dece sli~no virusnim hepatitisima.

Klju~ne re~i:

hroni~ne bolesti jetre, morfometrija, DNA

analiza, displazija.

Several studies have investigated the preneoplastic

changes in hepatocytes in chronic liver diseases in the

adults. Morphologic studies indicate that dysplasia is frequently

associated with the development of hepatocelullar

carcinoma (HCC) (1-4). Measurement of DNA content in

patients with chronic hepatitis have revealed a significant

correlation betwen the DNA index and the histological evidence

of HCC (5,6). Morphometric analysis of liver cells

have shown that increased nuclear area and/or nuclearcytoplasmatic

ratio represent the more likely candidate for

precancerous changes in the life (7,8).

Although these preneoplastic conditions have been

extensivelly studied in chronic liver diseases in adults,

similar studies in the paediatric age group are rare (1-8).

Available data consist of children with chronic HCV

(Hepatitis C virus) infection and they were primarily clinically

and morphologically oriented with no emphesis on

the premalignant changes (9-11).

Esquivel reviwed the histopathology of liver from 72

children with various causes of chronic liver diseases. He

detected 9 cases (12.5%) with HCC and/or liver cell dysplasia

(12). The primary liver diseases in these 9 children

was tyrosinemia (3), biliary atresia (2), chronic virus

hepatitis (2), neonatal hepatitis (1), and neonatal iron storage

disease (1). This sudy indicated that children with

chronic liver diseases may develop HCC early in the life or

show dysplastic changes which may proceed to carcinoma

later in the life.

The objective of the current study is to compare the

possible preneoplastic changes including dysplasia,

nuclear mophometric features and DNA abnormalities in

various form of chronic liver diseases in Egyptian children.

MATERIAL AND METHODS

Patients Seventy children diagnosed as having chronic

liver disease at the Paediatric Hospital, Cairo University,

Egypt were selected to constitute the subject of the current

study. Those children underwent careful clinical, radiological,

laboratory investigations and liver biopsy. Consequently,

they were classified into 3 groups. Table 1. The first group

was infantile cholestasis. It consisted of 30 children who were

diagnosed as neonatal hepatitis (19), paucity of bile ducts (8

cases), and extrahepatic biliary atresia (3 cases). The second

group included 24 patients with chronic hepatitis. Viral

hepatitis was confirmed in 9 of these patients (8 were hepatitis

C, 1 hepatitis B). Autoimmune hepatitis was diagnosed in

8; cryptogenic hepatitis in 7 patients. The third group consisted

of 16 children with glycogen storage disease.

Histopathological study True cut needle liver biopsies

were obtained from all children which enrolled this study.

They were fixed in buffered formalin and embedded in

paraffin. Tissue sections were stained with haematoxylin

and eosin (H&E), periodic acid Schill (PAS), and Massonís

trichrome stains. These sections were examined to confirm

the diagnosis, estimate the grade of disease activity, and

stage of fibrosis according to Scheuer (13). Moreover, the

presence of dysplasia was assessed as recommended by the

International Working Party (14).

DNA image anaysis Tissue sections from paraffinembedded

liver tissue were cut on poly-L-lysine coated

slides. These tissue sections were stained by Feulgen technique

using the quantitative DNA staining kit (Becton

Dickinson, Chicago,USA). Feulgen-stained slides were

analysed on the CAS200 image analyser (Becton Dickinson,

Chicago,USA), using quantitative DNA analysis software

programme. For each specimen, at least 200 non-overlapping

nuclei were analysed. Whenever possible, about 20

lymphocytes were also calculated to be used as an internal

diploid control. The DNA content was computed as the integrated

optical density of the Feulgen-stained nuclei (15).

DNA index (DI) of the studied tumours with the mean peak

was in the DI range of 0.9-1.1 (2C peak) or aneuploid

tumours with the DI fell outside the diploid range or more

than 20% of the cells were present in the G2/M phase (16).

DI was computed by dividing the model mass value of the

test cells by the known DNA content value of the control

cells in picograms (7.18pg) (17).

Morphometric analysis The nuclear features of liver

cells including nuclear area and nucleo-cytoplasmatic ratio

(N/C) were estimated by using H&E stained sections and

computerised image analyser (Cell Analysis System, Becton

Dickinson,Chicago,USA). For each case, the cell measurement

program ws applied to count randomly the nuclear features

of 300 hepatocytes at magnification x400. Then, the

mean nuclear area and mean N/C ratio were calculated.

Quantitative measurement of the severity of liver fibrosis

was achieved by using the same computerised image

analyser and Masonís trichrome-stained sections. The fibrotic

index (FI) was calculated for each case as the ratio of the

area of fibrosis to the area of the entire tissue specimen (18).

Statistical analysis Unpaired T-test was used to compare

the 3 categories of chronic liver diseases regarding

disease activity, stage of fibrosis, morphometric nuclear

features and fibrotic index. The 3 disease categories were

also compared with respect to dysplasia and DNA ploidy

by applying Chi-square test. Correlation between the grade

of disease activity and stage of fibrosis as well as between

the semiquantitative score of liver fibrosis (stage of fibrosis)

and the quantitative score of fibrosis (fibrotic index)

was assessed by Pearson,s correlation coefficient.

RESULTS

Histopathological data The group of infantile cholestasis

consisted of 30 children. Their liver biopsies were diagnosed

as neonatal hepatitis (19), paucity of bile ducts (8),

and extrahepatic biliary atresia (3). Evidence of cirrhosis

(stage 4) was found in 7 liver biopsies (23.3%). The group

of chronic hepatitis (CH) included 24 children. The aetiology

of hepatitis was viral in 9 cases (8 hepatitis C virus, 1

hepatitis B virus ), autoimmune (8), and cryptogenic (7).

Eleven out of 24 liver biopsies (45.8%) showed cirrhosis.

The last group in this study was glycogen storage diseases

(GSD) which consisted of 16 children. Eight of their liver

biopsies (50%) showed cirrhosis (Table 2).

The grade of disease activity was not significantly different

among the 3 disease groups. The stage of fibrosis

was significantly positive higher in the cholestatic group

than in GSD (T=2, p=0.05). A highly significant positive

correlation was obtained between the grade and stage

(r=0.37,p=0.002). Figure 1. The frequency of dysplasia

was significantly more in the cholestatic group (x2=10.8,

p<0.01) and chronic hepatitis (x2=14.5, p<0.01) than in

GSD. However, no significant difference was detected

between the former two groups.

DNA image analysis (Table 3) Aneuploid cell populations

were seen in 8 out of 70 paediatric cases with chronic

liver diseases (11.4%) with DNA index (DI) ranging

between 1.18 and 1.51 (mean 1.25). All these 8 biopsies

had the morphologic features of dysplasia

Four out of 8 aneuploid biopsies were the cholestatic

group (Figure 2) and the other 4 were of the hepatitis group

(Figure 3). All 16 liver specimens with GSD displayed the

classic diploid pattern (Figure 4) with minor variation for

the DI.

Morphometric anaysis The group of infantile cholestasis,

chronic hepatitis and GSD were not statistically different

regarding the nuclear area and the fibrotic index.

However, N/C ratio was significantly higher in the choelstatic

group (T=2.4, p=0.02) and in the hepatitis group

(T=3.3, p=0.003) than in the GSD group. There was a

highly significant correlation between the semiquantitative

(staging) and the quantitative (FI) estimation of fibrosis

(r=0.5, p=0.0001). Figure 5.

DISCUSSION

The commonest aetiology of chronic liver diseases in

this study of 70 Egyptian children with chronic liver diseases

is infantile cholestatic syndromes (43%). Conversely,

viral hepatitis accounted only 13% of the total cases. This

findings is consistent with North American, European, and

Indian reports which suggest that the incidence of chronic

hepatitis B and C is of lower frequency in childhood

(20,21). Our results are in parallel with other Egyptian

population based studies which indicate that chronic HCV

is much rarer below the age of 20 years (8%) (22).

Assessment of the grade of disease activity and stage of

fibrosis in the liver biopsies from children with viral

hepatitis revealed that although they demonstrate low

grades of disease activity, the stages of fibrosis are much

higher. This agrees with Badizadegan who suggested that

in spite of mild histological necroinflammatory activity,

the stage of fibrosis in children with HCV hepatitis can be

more pronounced (9).

In the study of the international childhood cancer incidence

of coordinated by the Agency for Research on

Cancer, hepatocelullar carcinoma (HCC) in children was

most common in parts of Asia and Africa including Egypt

(19).

The histological, morphometric and flow-cytometric

investigations are increasingly defining the preneoplastic

hepatic lesions and the stepwise evolution of HCC in

adults. However, the search for similar studies in children

are lacking. This might be related to presumed small incidence

of HCC in childhood and small number of well documented

cases (23,23,25). However, in the last few years

the incidence of HCC in children seems to be rising. This

necessitates selection of children with chronic liver diseases

who are prone to develop hepatic malignancies and

further on deserve careful follow up.

We find that the frequency of hepatocelullar dysplasia

is significantly higher in children with infantile cholestasis

and chronic hepatitis than in those with GSD. Since liver

cell dysplasia has been claimed to play a role in the evolution

of HCC, our results suggest that infantile cholestasis

and chronic hepatitis have a more tendency for malignant

transformation than GSD (1-4). This is supported by the

observation that dysplastic liver cells show aneuploid

DNA pattern as revealed in the current study and others

(2,5,6,29). It is worth to mention that the importance of

viral hepatitis as potential risk factor for malignancy has

been previously reported in several studies ( 8,30-33).

Similarly, the absence of dysplasia and aneuploidy in cases

of GSD is supported by the tendency of GSD to progress

to hepatocelullar adenoma rather than carcinoma (34).

Nevertheless, our observation that infantile cholestasis has

similar risk for developing malignancy as chronic hepatitis

has not been reported previously. This is to support comparable

increase in the N/C ratio in the cholestatic and

hepatitis groups which were significantly higher than N/C

ratio in the GSD group. The reported cases of HCC in children

with infantile cholestasis are only confined to cases

with familial intrehepatic cholestasis syndromes which

constitute about one fourth of the cases in the colestatic

group in this study (35-37). The majority of the children in

this group had neonatal hepatitis (NH) which was not an

antecedent of HCC in childhood. However, Moore recently

reported a child who was diagnosed as having NH at 4th

months of life, developed cirrhosis at 23rd months of age,

and HCC at 28th month (38). Thus, it is belived that NH

may be one of paediatric chronic liver disease tham may

eventually progress to HCC. Therefore, follow up of children

suffering of NH may be recommended especially in

those developing cirrhosis and/or dysplasia with aneuploid

DNA pattern. In fact, 4 out of 19 children included in this

study showed cirrhosis and another 2 children revealed

high grade of dysplasia with aneuploid cell populations in

their liver biopsies.

One of the important points in the current study is the

morphometric estimation of the fibrotic index. This quantitative

measurement of fibrosis was well correlated with

the semiquantitative measurement of the stage of fibrosis.

This finding whish is supported by several reports indicates

that image analysis is simple, rapid and reproducible

tool for the objective quantification of liver fibrosis (39-

41). Although the assessment of liver dysplasia, DNA

ploidy and N/C ratio revealed that these potential premalignant

changes were more in the cholestatic and chronic

hepatitis children than those with GSD, the fibrotic index

was not significantly different among our 3 groups. This

result may indicate that the degree of firbrosis in not necessarly

a measure of the preneoplastic potential of the

chronic liver disease. In the other words, cirrhosis might be

an essential event in the multistep evelution of HCC in

children as well. This is supported by stidy of Berman who

founded that cirrhosis is found in only 15% of HCC in children

(42). Yet, large studies are necessary to further define

this proposal.

Finally, the current preliminary study imply that chronic

viral hepatitis appears to constitute a minor percentage

of chronic liver diseases in Egyptian children. In order to

predict the prognosis and objectively measure liver fibrosis

degree, image analysis especially morphometry is an

important tool. Our stady further indicates that infantile

cholestasismay have some premalignant potential similar

to the chronic viral hepatitis. We found that liver cell dysplasia,

DNA aneupolidy and increased N/C ratio can be

used as risk factor for developing HCC in children with

chronic liver diseases. Conversely, the value of the fibrotic

index as indicator of high risk paediatric cases with liver

diseaes was not confirmed in this study.

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