tumor in the light of the revised SIOP-01 classification

ISSN 1427−4426
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Annals of Diagnostic Paediatric Pathology
Official Journal of the Polish Paediatric Pathology Society
and Section of Oncological Surgery of Polish Association of Paediatric Surgeons
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The Annals of Diagnostic Paediatric Pathology is an international peer−reviewed journal. The focus of the journal is current progress in
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© Copyright by Polish Paediatric Pathology Society, 2001
ISSN 1427-4426

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Annals of Diagnostic Paediatric Pathology
Official Journal of the Polish Paediatric Pathology Society
and Section of Oncological Surgery of Polish Association of Paediatric Surgeons
Volume 10 Number 3–4 Winter 2006
Review
paper
Original
papers
Case
report
CONTENTS
Pathobiology of arrhythmogenic right ventricular cardiomyopathy . . . . . . . . . . . . . . . . . . 65
El¿bieta Czarnowska, Bo¿ena Cukrowska
Is it possible to diagnose Hirschsprung’s disease and allied disorders on suction
rectal biopsy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Anna Piaseczna-Piotrowska
Treatment of malignant pheochromocytoma in children . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Jolanta Krajewska, Kornelia Hasse-Lazar, Barbara Jarz¹b
Paediatric differentiated thyroid cancer – differences in biology and treatment . . . . . . . . 81
Daria Handkiewicz-Junak, Barbara Jarz¹b
Blue Native Electrophoresis: an additional useful tool to study deficiencies
of mitochondrial respiratory chain complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Agnieszka Karkuciñska-Wiêckowska, Katarzyna Czajka, Micha³ Wasilewski, Jolanta Sykut-Cegielska,
Maciej Pronicki, Bo¿ena Cukrowska, Ewa Pronicka, Krzysztof Zab³ocki, Jerzy Duszyñski, Mariusz R. Wiêckowski
Comparison between the efficiency of hair follicle- and epidermal-derived keratynocyte
cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Tomasz Drewa, Bartosz Nadolski, Ilona Sir, Artur Czaplewski, Przemys³aw Ga³¹zka, Andrzej I. Prokurat
Histopathologic features of Wilms’ tumor in the light of the revised SIOP-01 classification
– one centre retrospective analysis of 44 cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Jerzy Niedzielski, Rafa³ Becht, Katarzyna Taran
Outcome of wide liver resections in children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Adam Bysiek, Joanna Palka, Andrzej Zaj¹c, Sabina Kantorowicz, Miros³aw Krysta, Bartosz Bogusz,
Wojciech Górecki, Anna Taczanowska-Niemczuk, Ma³gorzata Zamora
The study on the pathogenesis of intestinal dismotility and malabsorbtion in gastroschisis
on experimental model – chicken embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Hanna Bu³hak-Guz, Regina Cybulska, Ewa Czichos, Tomasz Krawczyk, Andrzej Kulig, Andrzej Chilarski
Humoral and cytokine responses induced by probiotic Lactobacillus casei and paracasei
strains in children with atopic dermatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Bo¿ena Cukrowska, Ilona Rosiak, Aldona Ceregra, Joanna Freszel, Gra¿yna Zakrzewska, El¿bieta Klewicka,
Ilona Motyl, Zdzis³awa Libudzisz
Cervical approach to anterior mediastinal tumors in children . . . . . . . . . . . . . . . . . . . . . . . 121
Miros³aw M. Krysta, Wojciech J. Górecki, Adam M. Bysiek, Witold H. Mie¿yñski, Krzysztof A. Solecki,
Bartosz J. Bogusz
Rare case of calcyfying fibrous pseudotumour of the lung in 6 years old girl . . . . . . . . . . . 125
Przemys³aw Przewratil, Anna Sitkiewicz, Józef Kobos, Ewa Andrzejewska
Malignant fibrous histiocytoma at 6 years old boy – a case report . . . . . . . . . . . . . . . . . . . . 129
Micha³ Rólski, Jerzy Harasymczuk, Przemys³aw Mañkowski, Andrzej Jankowski, Pawe³ Kroll,
Ma³gorzata Warzywoda, Ewa Trejster
Umbilical remnant abnormalities: a review of 5 cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Ma³gorzata Pacholska, Ma³gorzata Chrupek, Irena Daniluk-Matraœ, Przemys³aw Ga³¹zka, Magdalena Chrzanowska,
Roman KaŸmirczuk, Piotr Brzeziñski, Sylwia Drewa, Zdzis³aw Skok, Andrzej Igor Prokurat
Merosin deficient congenital muscle dystrophy in children – clinical features
and retrospective immunohistochemical study of own muscle biopsy material . . . . . . . . . 137
Maciej Pronicki, Hanna Mierzewska, Tamara Szymañska-Dêbiñska, Agnieszka Karkuciñska-Wiêckowska,
El¿bieta Karczmarewicz, Tomasz Kmieæ

64
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Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 65–69
© Copyright by Polish Paediatric Pathology Society Annals of
Pathobiology of arrhythmogenic right ventricular cardiomyopathy
El¿bieta Czarnowska, Bo¿ena Cukrowska
Department of Pathology
The Children's Memorial Health Institute
Warsaw, Poland
Arrhythmogenic right ventricle cardiomyopathy (ARVC) is
a myocardial disease characterized by replacement of
myocardium by fatty or fibro-fatty tissue, primarily in the right
ventricle (RV) and involvement of the left ventricle in nearly
half cases. [29]. The disease processes originate from
subepicardium and extend to the subendocardium, usually (in
80% cases) sparing ventricular septum [8].
ARVC familial occurrence with autosomal dominant
inheritance, variable expression and incomplete penetrance is
estimated from 1/2000 to 1/5000 [10]. The disease affects
men more frequently than women in ratio 3:1. Heredofamilial
background is found in about 50% of cases. Usually the
disease inheritance is autosomal dominant with incomplete
penetrance and variable expression. Till now, six of disease
genes (plakoglobin, desmoplakin, plakophilin-2, desmoglein-
2, RyR2, TGFß3) have been identified [4, 13, 22, 27, 29, 31].
The disease is at high risk of sudden death, particularly
in young people and athletes [9]. Pathogenesis of the disease
despite of genetic findings is still poorly known.
Clinical features
Abstract
Characteristic clinical abnormalities include delayed depolarization,
epsilon waves and inverted T waves in the right precordial
leads, late potentials, ventricular dilatation, systolic
Address for correspondence
Arrhythmogenic right ventricular cardiomyopathy is a disease characterised by replacement of mainly right
ventricular myocardium by fatty or fibro-fatty tissue but its pathomechanizm still not completely known.
In the review clinical features of the disease, histopathology of endomyocardial biopsy specimens, disease
pathobiology are presented, and pathomechanism in the context of genetic mutations is discussed.
Key words: arrhythmogenic right ventricular cardiomyopathy, anatomopathology, histopathology, gene
mutation, pathomechanism
El¿bieta Czarnowska tel. +22 8151971
Department of Pathology fax +22 8151975
The Children’s Memorial Health Institute e-mail: e.czarnowska@czd.pl
04-736 Warsaw
Al. Dzieci Polskich 20
Diagnostic
Paediatric
Pathology
and wall motion injury. Palpitation and syncope due to occurrence
of ventricular tachycardia of right ventricular origin,
with left bundle branch block are frequent. Electrical instability
may be triggered by exercise or stress. Standarized
diagnostic criteria of ARVC diagnosis are based on the presence
of 2 major or 1 major plus 2 minor or 4 minor criteria
established by an international task force [21], and shown in
Table 1.
It could be pointed that clinical presentation of the disease
is related to range of myocardial pathology and can
subdivided into three phases:
! Concealed phase without symptoms and minor electrical
instability; patients are frequently asymptomatic
but at risk of sudden death, particularly during intense
physical exertion;
! Overt phase with symptomatic ventricular arrhythmia,
patients present palpitation, and/or syncope,
morphological abnormalities can be detect by imaging
techniques (Echo, Magnetic Resonance);
! Advanced phase with sever myocardial loss causing
impairment contractility of the right ventricle [7].
In the advanced phase the disease may clinically mimic
dilated cardiomyopathy and in the end stage the left ventricle
abnormalities may be involved. It should be emphasized
that patients often present broader spectrum of disease

66
Table 1
Major and minor criteria for clinical diagnosis of ARVC. [adopted from Corrado et al. 2006]
MAJOR MINOR
Family history disease confirmed at necroscopy sudden death at 12year and absence
RBBB
■ sustained or nonsustained LBBB,
ventricular tachycardia
Documented in ECG or Holter,
extrasystoles (>1000/24h on Holter)
global or regional severe dilation and reductionof RV ejection mild global RV dilation or ejection fraction
dysfunction and fraction withwith no or mild LV involvement; reduction with normal LV; segmental
structural alterations localized RV aneurysms severe segmental
dilatation of RV
dilation of RV, regional hypokinesia
EMBs fibrofatty replacement of myocardium
Abbreviations: RBBB – right bundle branch block, LBBB – left bundle branch block, abN – abnormal, EMBs – endomyocardial
biopsies, ECG – electrocardiogram
and the phases mentioned above can be hardly recognized.
The disease expression is hardly diagnosed particularly at
early stages and in children, who did not yet develop full clinical
disease features.
Anatomopathological features
Two variants of ARVC hearts according to the nature of myocardium
replacement by fatty and fibrofatty tissue can be distinguished
(Fig. 1) [11]. This replacement occurs in a seg-
Fig. 1 Progressive fatty replacement of the myocardium in the right ventricle
wall (RV) with thinning of the RV wall
mental, patchy fashion. The consequences of ventricle wall
structural injury are diffuse hypokinesis or regional wall motion
abnormalities. The presence of fat and/or fibrosis in the
RV coexisted with the degenerative changes of the myocytes
trapped within area and their loss are adequate in diagnosis
of ARVC [8].
In the fatty variant diffuse (in 20% cases) or segmental
(80% cases) pattern of fatty localization in the RV is observed.
Moreover, septum is involved in 20% cases [2].
A marked decrease of myocardial cells associated with replacement
by fatty cells starts in the area called “triangle of dysplasia”
that is the right ventricle outflow tract, the apex and
the inferior wall. In this type of heart infiltration of inflammatory
cells is rarely observed.
The fibro-fatty variant hearts exhibit thickening of the
right ventricle wall < 3mm and inflammatory cell infiltration
in 2/3 of cases [2]. This infiltration may contribute to electrical
instability and sudden death and the disease progress
to heart failure. What is an etiologic agent triggering inflammatory
process remains not clear since the presence of cardiotropic
viruses is controversial [7]. Some investigators did
not find cardiotropic viruses [6] while other reported the presence
of retroviruses and adenoviruses in 59% cases of 12
sporadic ARVC [5,14].
Findings in endomyocardial biopsy samples
The histological findings of fatty or fibrofatty tissue in endomyocardial
biopsy (EMB) samples are not diagnostic per

se and they can be observed in myocarditis and other cardiomyopathies.
In ARVC fatty or fibrofatty tissue in EMBs
coexist with cardiomyocytes loss. Analysis of significant
number of biopsy samples made by Angelini et al. suggests
that the amount of fibro-fatty tissue exceeding 43% of the
samples together with loss of myocytes is diagnostic [1]
(Fig. 2). It has also been shown that EMBs analysis improves
diagnostic accuracy in ARVC, being more sensitive tool
than nuclear magnetic resonance (89% vs. 56%) [23]. It
must be however taken into consideration that analysis of
biopsy specimens can be negative at early stages of the disease
and in children due to segmental pattern of myocardial
replacement.
Fig. 2 Section of the right ventricle endomyocardial biopsy samples stained
with Trichrom. Microscopic magnification ×20. Adipose tissue – F,
myocytes – red staining, fibrous tissue – blue staining
A high cell death by apoptosis was reported in
ARVC, although there is discrepancy among investigators
about intensity of this process [20, 25, 32]. The apoptosis has
been discussed as potential mechanism of myocardial atrophy
and consequence of desmosomes injury.
Recent electron microscopical studies revealed abnormalities
of cellular junction located at intercalated disc, i.e.
desmosomes, fascia adherens and nexuses. In the past has been
noticed that couplings at intercalated disc are pale [15].
The recent morphometric analysis have shown the increase
of desmosome mean length and wideness of gap between cell
membrane in the area of desmosmes and fascia adherens
(Fig. 3A), and presence of abnormally short junctions composed
of series reaping couplings (found in 11 out of 21 cases)
(Fig. 3B), and decreased number of nexuses per 10 m
cell membrane unit [3]. No statistically significant differences
among the ultrastructural features have been found between
cases with diagnosed gene mutation and genotype negative
patients [3].
F
F
F
Fig. 3 Cardiomyocyte couplings at intercalated disc. A) “Pale” contrast of
junctions and long desmosome in this area (arrow). Microscopic
magnification ×10 000. B) Region of the junctions with series of short
desmosomes (arrows). Microscopic magnification ×30 000
Genetic
ARVC is mostly inherited in autosomal dominant fashion but
recessive variant called Naxos disease is also known. Ten types
of disease are known (Table 2).
Naxos disease is characterized by common heart abnormalities
coexisted with palmoplantar keratoderma and
wool hair. Two base pair deletion in the plakoglobine gene
has been identified [21]. Plakoglobin is one of major constituent
of desmosmes.
Table 2
ARVC types and genetics. D – autosomal dominant, R – autosomal
recessive, TGF-ß3 – transforming growth factor
beta-3, RyR2 – ryanodine receptor 2, DSP – desmoplakine,
PKP2 – plakophilin 2, DSG2 – desmoglein 2, PKG – plakoglobin
Disease Chromosome Gene Mode of
type locus defect transmission
AKPK1 14q24.3 TGF- 3 D
AKPK2 1q42-q43 RyR2 D
AKPK3 14q12-q22 ? D
AKPK4 2q32.1-q32.3 ? D
AKPK5 3p23 ? D
AKPK6 10p12-p14 ? D
AKPK7 10q22.3 ? D
AKPK8 6p24 DSP D
AKPK9 PKP2 D
AKPK10 18q12,1 DSG2 D
Naxos disease 17q21 PKG R
67

68
Among gene linked to autosomal dominant ARVC
were found genes coding other than plakoglobin proteins related
to mechanical junctions, i.e. desmoplakin [28], plakophilin-2
[13], desmoglein-2 [27]. Desmoplakin together
with plakoglobin anchores to cadherins, desmoglein or desmocolin,
which comprise the transmembrane component of
desmosome. Plakophilin is located in the outer dense plaque
of desmosomes and links cadherins to desmoplakine. Because
these four proteins are involved in ARVC the disease
is considered as a disease of the desmosomes. Desmosomes
proteins are linked to actin network while fascia adherens
proteins to intermediate filaments e.g. desmin network. Therefore,
abnormalities in proteins of both desmosomes and fascia
adherens might lead to impairment of cell-cell contact
[17]. Impaired cell mechanical coupling may predispose cardiomyocytes
to detachment and death under conditions of
mechanical stress. Destabilization of cell-cell adhesion
complex also disturb normal turnover of conexines, the element
of gap junction (nexuses), which play a role in electrical
cell coupling. Diminished expression of connexin 43 at
the intercalated disc has been also observed in patients with
Naxos disease [16]. This feature in the context of the role of
nexuses in ion transport and transfer of electrical stimulation
may result in heterogeneous electrical conduction and arrhythmia.
Other found genes linked to autosomal dominant
ARVC are cardiac ryanodine receptor 2 (RyR2) and transforming
growth factor beta-3 (TGF -3). Mutations in gene RyR2
results from substitution of amino acids in domains which
are critical for regulation of the calcium channel. Up to present
mutations in RyR2 association with cardiac diseases beside
of ARVC 2 has been also found in catecholaminergic
polymorphic ventricular tachycardia [28] and familial poly-
morphic ventricular tachycardia diseases [18]. These diseases
and ARVC are characterized by effort-induced polymorphic
ventricular arrhythmias and a risk of sudden death.
These might be due to fact that mutation in RyR2 alter the
ability of calcium channel to remains closed and thus stress
or physical effort initiate intercellular calcium overload leading
to severe arrhythmias.
In affected probants of ARVC type 1 a nucleotide
substitution in 5’UTR and in one subject substitution in
3’UTR in the TGF-ß3 gene were detected [4]. It is known
that transforming growth factor of TGF-ß family inhibits
proliferation of many type of cells and stimulate mesenchymal
cells to proliferate and promote fibrosis [19].
Pathomechanism
In the context of defects in intercellular junction of cardiomyocytes
it suggested that volume overload in the right ventricle
would produce myocardial over-stretch. It is known
that stretched cardiomyocytes exhibit abnormal release of
calcium from ryanodine receptor channel [26] what affects
intracellular calcium concentration. Volume overload in
subjects, carriers of mutation in RYR2 can also lead to calcium
overload. Both disconnection of cellular junctions and
calcium overload can be also related to increased cardiomyocyte
apoptosis (Fig. 5). It has been suggested that fibrofatty
replacement is repair process. However recently Garcia-
-Gras et al. (2006) exploring the effects desmoplakine deficiency
on Wnt/ß catenin signaling found that translocation
of plakoglobin into cardiomyocyte nuclei led to up-regulation
of genes implicated in adipogenesis [12]. This signaling
pathway could clear increased quantity of fat in ARVC
hearts.
Fig. 4 Molecular model of adherens junction and desmosome organization Fig. 5 Proposed patomechanism of ARVC

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(2004) Mutations in the desmosomal
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(2005) Induced deletion of the N-cadherin
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(2001) Mutations of the cardiac ryanodine
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Circulation 103: 485–490
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(1996) Evidence of apoptosis in arrhythmogenic
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(1994) Diagnosis of arrhythmogenic right
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of the International Society
and Federation of Cardiology. Br Heart
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et al. (2000) Identification of a deletion
in plakoglobin in arrhythmogenic right
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keratoderma and woolly hair
(Naxos disease). Lancet 355:
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(1996) Spin-echo nuclear magnetic resonance
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arrhythmogenic right ventricular cardiomyopathy.
Heart 76: 467–470
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Clinical profile and long-term followup
of 37 families with arrhythmogenic
right ventricular cardiomyopathy. J Am
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et al. (1999) Programmed cell death in
the myocardium of arrhythmogenic right
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(2001) Endogenous nitric oxide mechanisms
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(2006) Mutations in desmoglein-2 gene
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Colombi B, et al. (2002) Clinical and
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M, et al. (1996) Report of the 1995
WHO/ISFC task force on the definition
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31. Tiso N., Stephan D. A., Nava A., et al.
(2001) Identification of mutation in the
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32. Valente M., Calabrese F., Thiene G, et
al. (1998) In vivo evidence of apoptosis
in arrhythmogenic right ventricular cardiomyopathy.
Am J Pathol 152: 479–84

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 71–74
© Copyright by Polish Paediatric Pathology Society Annals of
Is it possible to diagnose Hirschsprung's disease
and allied disorders on suction rectal biopsy?
Anna Piaseczna-Piotrowska
Departament of Pediatric Surgery and Urology
Polish Mother's Health Institute
Lodz, Poland
Abstract
Our knowledge of the pathomorphology of Hirschsprung’s
disease (HD) and allied disorders, especially intestinal neuronal
dysplasia (IND) and hypoganglionosis is mainly based
on information derived from histological and histochemical
examinations. The diagnosis of HD and allied disorders can
be established by full-thickness rectal biopsy and evaluation
of the myenteric plexus, but the easier and less invasive suction
rectal biopsies (SRB) have superseded this procedure.
SRB gives excellent results in excluding the diagnosis of
HD, although positive diagnosis of HD requires confirmation
by full thickness biopsy usually at the time of colostomy or
pull through operation.
Obtaining optimal suction biopsy specimens to confirm
or rule out HD or allied disorders requires good equipment
and a meticulous technique. SRB sections should be taken
at 3, 5, and 7 cm just above the pectinate line. The biopsy
at 7 cm is omitted in neonates [6, 10, 14, 18, 20, 23]. It is
important that the submucosa is included in the specimen. An
adequate biopsy is 3 to 4 mm in diameter and 1 to 2 mm deep
[10, 26]. Small size of sample with a sparse distribution of
submucosal ganglia cells in the normal gut may lead to an
erroneous diagnosis of HD or allied disorders. Nonetheless,
Address for correspondence
The introduction of suction rectal biopsy (SRB) while making the procedure less traumatic for the patient
has made the diagnosis of Hirschsprung’s disease (HD) and allied disorders more difficult for the
pathologist. The development of histochemical techniques for the detection of acetylocholinesterase
(AChE) and nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d) was a considerable
advance in the diagnosis of this disorders. The aim of this study was to evaluate the results of AChE and
NADPH-d staining in SRBs of patients presenting with chronic constipation or dysmotility problems.
Key words: enzyme histochemistry, Hirschsprung’s disease, hypoganglionosis, intestinal neuronal
dysplasia, suction rectal biopsy
Anna Piaseczna-Piotrowska Tel. No: 0 48 42 271 21 36
Polish Mother’s Health Institute Fax No: 0 48 42 271 13 58
Rzgowska 281/289 E-mail: annapiaseczna@yahoo.com
93-322 Lodz, Poland
Diagnostic
Paediatric
Pathology
unless a patient has HD, ganglion cells are almost always encountered
if more than 50–75 sections are examined from an
adequate biopsy [26]. Only 55–65% of the sections with vertical
cuts of mucosa show nerve ganglia. The yield of nerve
ganglia cells is much higher in biopsies larger then 5 mm cut
parallel to the mucosal surface [10, 16, 18, 21].
The interpretation of morphological findings in SRB
specimens is often difficult and in particular dependent on
the staining methods used. Considerable experience is required
to obtain a reliable diagnosis if hematoxylin and eosin
(H&E) staining is used. Hypoganglionosis and ultrashort HD
can be diagnosed wrong. The introduction of new histochemistry
methods either AChE reaction in RSB specimens had
made a morphological diagnosis easier and more reliable
[24]. AChE is especially expressed in nerve fibres and ganglia
cells [7, 9, 15, 24]. Nitric oxide (NO) has recently been
recognised as a non adrenergic, non cholinergic neurotransmitter
that mediates relaxation of the smooth muscle of the
gastrointestinal tract (GIT). Histochemical studies have
shown that NO-synthase is identical to NADPH-d. Several
investigators have demonstrated, that NADPH-d activity is
markedly altered in HD and allied disorders [20, 23–25].

72
Fig. 1 SRB in normal bowel. (A) AChE staining shows few AChE-positive
nerve fibres present in the LP and MM. Ganglia cells are visible in the
submucosal layer. (B) NADPH-d histochemistry shows strong activity in
submucosal ganglia cells and large numbers of NADPH-d positive nerve
fibres in MM
Normal SRB specimens contain only a few fine cholinergic
nerves in the lamina propria (LP) (Fig. 1). In the normal
muscularis mucosa (MM) a small number of nerve fibres
are always present. Ganglia cells in the submucosal layer are
evident [1, 3, 9, 14, 21, 26].
The histochemical diagnosis of HD is based on the
fact that the cholinergic nerve fibers of the aganglionic segment
are prominent and that these fibres contain an increased
of amount of AChE (Fig. 2) [1, 7, 9, 15, 21]. Three AChE
histochemistry patterns of the rectal suction biopsy are established
[1, 3, 15]:
Newborn pattern: AChE- positive thick nerve trunks
or coarse nerve fibres are present in submucosa layer. Characteristically
there is absence of AChE activity in the LP and
weak activity in MM. This pattern is predominant in infants
up to 3 months.
Fig. 2 SRB in HD (A) AChE-positive nerve fibres are markedly increased
in the LP and MM. Ganglia cells are absent in submucosae. (B) NADPH-d
activity in the MM is completely absent and hypertrophic nerve trunks stain
very weak. No ganglia cells can be found
Classic pattern: Increased AChE positive thin nerve
fibres are present in the MM with a clear infiltration in the
LP. This pattern is mainly seen in children older then 1 year.
Intermedite pattern: This pattern not related to age.
It shows AChE positive nerve fibres in the three layers of
SRB specimens but in one or more layers the nerve fibres are
spare.
There is no evidence of ganglia cells in all patterns.
In total colonic aganglionosis the changes may appear
particularly mild with only a few nerve trunks in the submucosa.
There may be no increase of AChE positive nerve
fibres in the LP and only a mild to moderate increase in the
MM [9].
The lack or marked deficiency of NO-synthase containing
nerve fibres in aganglionic colon was reported by several
investigators. NADPH-d activity in the MM is completely
absent and hypertrophic nerve trunks stain very weak.
There is no evidence of ganglia cells in the submucosa [20].
The histological criteria of IND keep changing. In
Meier’s-Ruge original description (1971), hyperplasia of
ganglia cells and increased AChE activity in parasympathetic
nerve fibres in the LP were regarded as the most important
diagnostic criteria [5, 11, 23–25]. In 1995, Borchard and
Meier-Ruge presented guidelines for identifying IND in
SRB. These comprised two obligatory criteria: hyperplasia of
submucosus plexus and an increase in AChE -positive nerve
fibres around the submucosal blood vessels, and two additional
criteria: neuronal heterotophia and increased AChE activity
in the LP [4, 23, 24, 25]. More recently, Meier-Ruge et
al, described hyperganglionosis and increased AChE activity
as age-dependent findings and giant ganglia as the most
characteristic diagnostic feature (although this can be a normal
finding in neonates) [5, 17, 23, 24]. Submucosal giant
ganglia (containing more than 7 nerve cells) comprise only
3–5% of all ganglia in IND and are usually not seen within
6–7 cm above the pectinate line [5, 8, 16, 17, 19, 23, 25].
In summary, histochemical diagnosis of IND in SRB
is very difficult. Some characteristic features suggesting the
presence of this disorder are hyperganglionosis of the submucosus
plexus, giant ganglia, ectopic ganglia, increased
AChE-positive nerve fibres around submucosal blood vessels
and increased AChE-positive nerve fibres in LP (Fig. 3)
[2, 5, 10, 12, 13, 17, 18, 23, 24, 27–29].
The diagnosis of hypoganglionosis in SRB usually is
very difficult to establish SRB specimens from these patients
show absence of submucosal ganglia cells or small single
ganglia cells and very scant activity in LP and MM (Fig. 4).
Full thickness rectal biopsy and morphometric studies are essential
to confirm this diagnosis [22–24].
General agreement exists on the diagnosis of HD on
the basis of SRB specimens, but controversy exists regarding
the identification of different innervation disorders based
upon SRB containing only the part of the Meissner’s plexus
[15, 20]. Many biopsies are disappointing because only superficial
mucosal fragments without submucosa and MM are
obtained. The histochemical diagnosis of IND and hypoganglionosis
in adequate SRBs is particularly difficult. The use

Fig. 3 SRB in IND. (A) AChE staining shows hyperplasia of the submucosal
plexus and giant submucosal ganglia, marked increase AChE-positive nerve
fibres around blood vessels. (B) NADPH-d positive nerve fibres in MM are
usually moderate. Submucosal giant ganglia stain strongly
of two different histochemical staining methods i.e. AChE
and NADPH-d may facilitate diagnosis of these disorders [4,
5, 11, 13, 14, 16, 20].
In conclusion, histochemical staining for the detection
of AChE and NADPH-d activity in SRBs is a simple and reliable
method for the diagnosis of Hirschsprung’s disease, in-
References
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Evolution with age of the acetylcholinesterase
activity in rectal suction biopsy
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2. Berry CL (1993) Intestinal neuronal
dysplasia: does it exist or has it been invented?
Virchows Arch A Pathol Anat
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3. Chentanez V, Chittmittrapap S, Cheepsoonthorn
P, Agthong S (2000) New
classification of histochemical staining
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1196–1201
4. Cord-Udy CL,. Smith VV, Ahmed S,
Risdon RA Milla PJ (1997) An evaluation
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5. Gillick J, Tazawa H, Puri P (2001) Intestinal
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treatment in 33 patients. J Pediatr Surg
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T, Taguchi T, Suita S (1993) The
simple technique of rectal mucosal
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942–944
7. Holschneider AM, Meier-Ruge W, Ure
BM (1994) Hirschrung’s disease and allied
disorders-a review. Eur J Pediatr
Surg 4: 260–266
8. Holschneider AM,. Puri P (2000) Intestinal
neuronal dysplasia. In: Holschneider
AM, Puri P (eds), Hirschsprung’s
disease and allied Disorders. Harwood
Academic Publishers, pp 147–153
9. Howard ER (1972) Hirschsprung’s disease:
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physiology. Postgraduate Med J: 48:
471–477
10. Kapur RP (1999) Hirschsprung’s disease
and other enteric dysganglionoses.
Crit Rev Cl Lab Sci 36: 225–273
11. Kobayashi H, Hirakawa H, Puri P
(1996) What are the diagnostic criteria
for intestinal neuronal dysplasia? Pediatr
Surg Int 10: 459–464
Fig. 4 SRB in hypoganglionosis. (A) AChE staining shows absence ganglia
cells with no or extremely weak AChE activity in the MM and LP. (B)
NADPH-d histochemistry shows a few NADPH-d positive nerve fibres in
the MM and absent submucosal ganglia cells
testinal neuronal dysplasia and hypoganglionosis. SRB is
a useful and valuable method to confirm or exclude Hirschsprung’s
disease. It is of little value in the recognition of IND
and hypoganglionosis. Presence of these neuronal intestinal
disorders should be confirmed by full thickness biopsies and
morphological study.
73
12. Koletzko S, Bullauff A, Hadziselimovic
F, Enck P (1993) Is histological diagnosis
of intestinal dysplasia related to clinical
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children? Results of a pilot study.
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13. Koletzko S, Jesch J, Faus-Kebler T, et
al (1999) Rectal biopsy for diagnosis of
intestinal neuronal dysplasia in children:
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interobserver variation and clinical outcome.
Gut 9: 853–861
14. Lake BD, Puri P, Nixon HH, Nixon
HH, Claireaux AE, (1978) Hirschsprung’s
disease. An appraisal of histochemically
demonstrated acetylcholinesterase
activity in suction rectal biopsy
specimens as an aid to diagnosis.
Arch Pathol Lab Med 102: 244–247
15. Meier-Ruge W (2000) Histological
Diagnosis and Differential Diagnosis.
Holschneider AM, Puri P (ed): Hirschsprung’s
Disease and Allied Disorders.
Harwood Academic Publishers, pp
252–265

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16. Meier-Ruge WA, Brönnimann PB,
Gambazzi F, Schmid PC, Schmidt CP,
Stoss F (1995) Histopathological criteria
for intestinal neuronal dysplasia of
the submucosal plexus (type B). Virchows
Arch 426: 549–556
17. Meier-Ruge WA, Gambazzi F, Käufeler
RE, Schmid P, Schmidt ChP (1994)
The Neuropathological Diagnosis of
Neuronal Intestinal Dysplasia (NID B).
Eur J Pediatr. Surg 4: 267–273
18. Meier-Ruge WA, Schärli AF, Stoss F
(1995) How to improve histopathological
results in the biopsy diagnosis of gut
dysganglionosis. A methodological review.
Pediatr Surg Int 10: 454–458
19. Meier-Ruge WA, Schmidt PC, Stoss F
(1995) Intestinal neuronal dysplasia and
its morphometric evidences. Pediatr
Surg Int 10: 447–453
20. Miyazaki E, Ohshiro K, Puri P (1998)
NADPH-diaphorase histochemical staining
of suction rectal biopsies in the
diagnosis of Hirschsprung’s disease and
allied disorders. Pediatr Surg Int 13:
464–467
21. Monfore-Munoz H, Gonzales-Gomez I,
Rowland JM, Landing BH (1998) Increased
submucosal nerve trunk caliber
in aganglionosis. A “positive” and objective
finding in suction biopsies and
segmental resections in Hirschsprung’s
disease. Arch Pathol Lab Med 122:
721–725
22. Munakata K, Okabe I, Morita K, (1985)
Clinical and histological studies of neuronal
intestinal dysplasia. J Pediatr Surg
20: 231–235
23. Puri P (1997) Variants of Hirschsprung’s
disease. J Pediatr Surg 32:
149–157
24. Puri P (1998) Preface of Hirschsprung’s
disease and related disorders – recent
progress. Semin Pediatr Surg 7:
137–139
25. Puri P, Wester T (1998) Intestinal neuronal
dysplasia. Semin Pediatr Surg 7:
181–186
26. Qualman SJ, Pysher T, Schauer G
(1997) Hirschsprung’s disease: differential
diagnosis and seguelae. In:
Dahms BB, Qualman SJ (eds) Gastrointestinal
disease (Perspectives in Pediatric
Pathology, Vol 20). S. Karger AG,
Basel (Switzerland) pp: 111–126
27. Schärli AF (1992) Neuronal intestinal
dysplasia. Pediatr Surg Int 2: 2–7
28. Schärli AF, Meier-Ruge W (1981) Localized
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neuronal dyspasia. J Pediatr Gastr
Nutr 12 (2): 182–189

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 75–79
© Copyright by Polish Paediatric Pathology Society Annals of
Treatment of malignant pheochromocytoma in children
Jolanta Krajewska, Kornelia Hasse-Lazar, Barbara Jarz¹b
Nuclear Medicine and Endocrine Oncology Department
M. Sklodowska−Curie Memorial Cancer Center
and Institute of Oncology, Gliwice Branch
Gliwice, Poland
Introduction
Abstract
Pheochromocytoma and paraganglioma are unusual neoplasms
in children. Both tumors develop from chromaffin
cells and produce catecholamines including norepinephrine,
epinephrine and dopamine. Tumors that derive from the adrenal
medulla are defined as pheochromocytomas, whereas
extraadrenal tumors are named paragangliomas [4, 22, 23].
Pheochromocytoma is diagnosed in approximately
1% of pediatric hypertensive patients. About 20% of these
tumors occur in children, mostly in preadolescent boys and
teenage girls with an incidence of 2 per million [5]. The
most common symptoms in children are: hypertension, headaches,
palpitations, sweating and nausea [2, 22]. Blood
pressure elevation is rather permanent in contrast to adults
who develop paroxysmal hypertension in 30% of cases [2].
It is important to differentiate between paragangliomas and
neuroblastomas, the last more common in pediatric population,
because of their similarity in location, histologic features
and ability to secrete hormones [5, 22]. Neuroblastomas
usually are found in retroperitoneum, origin from neural
crest cells and produce vanillylmandelic acid (VMA) and
Address for correspondence
Pheochromocytoma is a rare neoplasm in children with the great difficulties in distinction between benign
and malignant tumors on the basis of histopathological examination. The only unequivocal evidence of
malignancy are distant metastases in sites where chromaffin tissue is absent. Although the treatment of
choice is radical surgical resection of primary tumor and metastases the adjuvant therapy is necessary.
131 I-MIBG therapy is the first-line systemic treatment. Somatostatin analogues labeled with 90 Y make an
alternative therapeutic option for patients with no 131 I-MIBG uptake. Chemotherapy, based on
cyclophosphamide, vincristine and dacarbazine is reserved for patients with recurrent and progressive
disease or without satisfactory response to previous treatment.
Key words: 131 I MIBG therapy, malignant pheochromocytoma, treatment
Diagnostic
Paediatric
Pathology
homovanillic acid (HVA). Catecholamines are produced
rarely.
Approximately 25% of pheochromocytomas are associated
with hereditary syndromes [21]. Genetic predisposition
is found as von Hippel-Lindau disease due to VHL gene
mutations, multiple endocrine neoplasia type 2A and 2B
(MEN) due to RET protooncogene mutations, in familiar paraganglioma/pheochromocytoma
syndromes associated with
mutations of succinate dehydrogenase family genes (SDHB,
SDHC and SDHD) and neurofibromatosis type 1 (NF1) due
to NF gene mutations [12, 21]. RET and VHL-dependent pheochromocytomas
are almost exclusively benign. The risk of
malignancy is the highest in SDHB mutation carriers [12].
The risk of malignancy reported in the literature on
adult population is about 10% but it ranges between of
5–26% depending on malignancy definition and population
investigated [6, 10, 12, 28]. The 5-year survival rate of patients
with malignant pheochromocytoma varies between
34–60% [2, 4, 18, 19, 24, 28]. However, more than 20-year
survival was also observed [2, 4, 28]. Children more often
have bilateral, multiple or extraadrenal tumors [2, 5, 16, 23].
Similarly, malignant tumors, although very rare, occur more
Jolanta Krajewska MD Tel: +48 32 2789301, fax +48 32 278 93 25
Nuclear Medicine and Endocrine Oncology Department E-mail: jkrajewska@io.gliwice.pl
M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology
Wybrze¿e AK 15
44-101 Gliwice

76
frequent in pediatric population. Ciftci et al diagnosed malignant
pheochromocytoma in 19% of cases [5] whereas in one
of the largest case series on pheochromocytoma and paraganglioma
in children from Mayo Clinic the malignancy rate
was 47% [23]. Five- and 10-year disease-specific survival in
the last group was 78% and 31% respectively [23].
The unequivocal distinction between benign and malignant
tumors on the basis of histopathological examination
is not possible. The only evidence of malignancy are the distant
metastases in sites where normal chromaffin tissue is
absent. They localize mostly in bones (44%), lymph nodes
and liver (37%) and lungs (27%) [19]. However, there are
a few prognostic factors that can suggest malignancy, such
as: tumor size larger than 5–6 cm, extraadrenal localization,
local invasion at the time of surgery and abnormal DNA ploidy
(aneuploidy and tetraploidy) [6, 10, 12, 23]. Some data
report that inhibin or activin β subunit expression which is
weak or absent in malignant tumors may predict malignancy
[4]. Tumor necrosis, nuclear atypia, vascular or capsular
invasion and mitotic index are not useful to distinguish benign
and malignant tumors [28].
Successful treatment of malignant pheochromocytoma
requires a multidisciplinary approach and is based on surgery,
targeted radiotherapy with 131 I-MIBG, chemotherapy
and pharmacotherapy.
Surgery
Radical surgical resection of primary tumor and metastases
after a period of pharmacological α-adrenergic blockade is
the treatment of choice [5, 6, 18, 19, 22, 23, 27, 28]. However,
in patients with disseminated disease radical surgical approach
is often not possible. Even palliative surgery to reduce
tumor burden can improve the response to further therapy
[18, 28]. Most operations are performed as open
laparotomy with full abdomen examination including contralateral
adrenal gland, lymph nodes and other known sites of
extraadrenal tumors [4, 12, 13, 16, 22, 27]. During the last
years minimally invasive surgery has been widely applied
and now tumor can be removed safely and successfully by
laparoscopic approach [4, 10, 13, 27]. However, tumors greater
than 8 cm should not be operated this way due to increased
risk of malignancy [27]. Tumor resection can lead to serious
complications because of the excessive release of catecholamines
and to postoperative hemodynamic and
metabolic disturbances as a result of sudden decrease in catecholamine
levels [2]. There are no significant differences
in blood pressure and heart rate between laparoscopic and
open surgery [22]. To lower blood pressure and avoid hypertensive
crisis patients should be pretreated pharmacologically
using α-blockade [2–5, 10, 12, 13, 22, 27, 28]. Phenoxybenzamine
hydrochloride (Dibenzyline) is a non-specific
β-adrenergic receptor antagonist. The most frequent side effects
are orthostatic hypotension, tachycardia and prolonged
hypotension after tumor removal [2–4]. To circumvent these
disadvantages selective α 1 -receptor antagonists such as
prazosin, terazosin or doxazosin can be applied [2–4]. In ca-
se of cardiac arrhythmias or tachycardia β-adrenergic receptor
antagonists (propranolol or cardioselective) or lidocaine
should be used [2, 12]. β-receptor blockers must not be given
alone or started before α-receptor antagonist due to possibility
of paradoxical increase in blood pressure accompanied
by heart failure and pulmonary oedema [2, 28]. However,
even if pharmacological treatment is sufficient it may
not prevent severe intraoperative hypertension, tachycardia
and ventricular arrhythmias [2–4, 10, 27]. The adequate preoperative
preparation criteria are given in table 1. Patients survival
rates range from 97,7–100% and residual hypertension
is observed in 27% to 38% cases [22].
Table 1
Criteria of adequate preoperative pharmacological preparation
[2]
1. Supine arterial pressure ≤160/90 mmHg
2. Orthostatic hypotension not exceeding 80/45 mmHg
3. ECG: ST segment or T wave free of changes for at
least 2 weeks
4. ≤ 1 ventricular ectopic beat every 5 min.
Radiopharmaceuticals
The first-line systemic treatment of malignant pheochromocytoma
is targeted radiotherapy. Since 1983 many centers
have reported successful use of metaiodobenzylguanidine labeled
with 131 I ( 131 I-MIBG) [6, 11, 12, 17-20, 24, 25, 28].
MIBG is a structural analogue of guanetidine, actively taken
up and stored in sympathetic neurons and related cells similarly
to norepinephrine [14, 17, 18]. Cellular MIBG uptake
is connected with two mechanisms: active uptake type I and
passive diffusion [18]. Accumulation and retention of
131 I-MIBG in tumor cells depends on expression of catecholamine
transporters on cell surface and in cytoplasmatic granules,
where catecholamines are stored [6]. The mechanism
of cytotoxic action is based on the emission of beta radiation
from decaying radionuclide 131 I [7]. 131 I-MIBG therapy is applied
in patients with positive diagnostic 131 I-MIBG whole
body scintigraphy confirming pathological focal uptake in
metastatic lesions. Life expectancy more than one month is
desired [7]. 131 I-MIBG is administered as a slow intravenous
infusion (30 min – 4 h). Single therapeutic dose ranges between
70–300 mCi (2,6–11,2 GBq), usually dose is 200 mCi
(7,4GBq), with cumulative activity between 200–1800 mCi
(7,4 – 66,6 GBq) [6, 7, 11, 17–20, 25, 28]. To prevent thyroidal
uptake and hypothyroidism potassium iodate (170 mg)
or potassium iodide (100–200 mg) should be given orally
48 h before 131 I-MIBG administration and continued for up
to 10 days post therapy. The usual dosing in adults is 3 × 20
drops of Lugol’s solution. The medicaments which may influence
on uptake or/and retention of 131 I-MIBG have to be
withdrawn for 1–2 weeks before treatment. These drugs are
presented in table 2 [7, 11, 17].

Table 2
The drugs which may interfere with the uptake and/or
retention of 131 I MIBG [7]
Proved interference Probable interference
Labetalol Adrenergic blockers:
bretylium, guanethidine
Reserpine Sympathomimetics:
Amphetamine, dopamine,
isoprenaline, terbutaline
Calcium channel Phenothiazines:
blockers chlorpromazine, promethazine
Tricyclic antidepressant: Butyrophenones:
Amitriptyline, imipramine droperidol, haloperidol
Sympathomimetics: Thioxanthines
ephedrine
Cocaine
maprotiline, trazodone
131 I-MIBG therapy is usually well-tolerated with minimal
side-effects. Nausea and vomiting may be present during
48 h after 131 I-MIBG administration [7, 19, 28]. Hypertensive
crises occur rarely, are caused by release of catecholamines
from tumor and require α-blockade. Mild bone marrow suppression
is typically observed 4–6 weeks post therapy especially
in patients with bone marrow involvement or with impaired
renal function. The limiting factor for this treatment is
cumulative bone marrow toxicity with persistent hematological
effects connected with myelosuppression. Hypothyroidism
may develop in some patients after inadequate thyroid blockade.
The risk of secondary cancer should also be considered.
Garaventa et al described 119 children with neuroblastoma treated
with 131 I-MIBG and chemotherapy. In 5 cases of study patients
second malignant neoplasms were observed, in particular
two cases of myeloid leukemia, one of malignant schwannoma,
one of angiomatous fibrous histiocytoma and one of
rhabdomyosarcoma. The authors emphasize the importance of
long-term follow-up in children treated with this approach [9].
131 I-MIBG therapy leads to 30–58% objective responses
defined as a reduction of tumor size and/or hormone levels
greater than 50% [12, 28]. Moreover, 50–75% of patients
show essential improvement in general condition and
quality of life [28]. Mukherjee et al reported a series of 37
patients with neuroendocrine tumors (15 with malignant pheochromocytoma
or paraganglioma) treated with 131 I-MIBG.
The mean single dose was 189 mCi (range 70–300 mCi) and
mean cumulative activity was 592 mCi (range 200–1592
mCi). None of the patients showed a complete remission.
Partial or complete symptomatic improvement was noticed
in 100% of cases. 89% of patients had a complete or partial
hormonal response and in 53% tumor reduction was observed
[20]. Lam et al also confirmed complete symptomatic
and complete or partial biochemical improvement in two patients
with repeated 131 I-MIBG therapy with high cumulati-
ve activity (1000 mCi and 1800 mCi). The disease has been
stabilized for several years with no serious side-effect of this
treatment [18]. Similarly, good palliative effect of 131 I-MIBG
therapy is reported by Hoefnagel and Konings [11, 17]. Rose
et al described 12 patients treated with high-dose
131 I-MIBG therapy with median single activity 800 mCi (range
386–866 mCi) and median cumulative dose 1015 mCi
(range 386–1690 mCi). 25% of them achieved complete remission
that has been sustained with up to 8 years of follow-up.
Two of these patients had bone and soft tissue metastases.
Moreover, 70% of patients showed symptomatic and
hormonal response. In 42% stable disease was stated. Grade
3–4 thrombocytopenia and neutropenia were noticed after
80% of treatments in this study and usually were transient.
However, the authors did not observe a correlation between
dose (mCi/kg) and hematologic toxicity [24].
Somatostatin analogues labeled with 90 Y make an alternative
therapeutic option for patients with no 131 I-MIBG
uptake or without favorable result of 131 I-MIBG treatment [6,
12, 28]. However, the expression of somatostatin receptors
on surface of pheochromocytoma tumor cells is lower than
on gastrointestinal endocrine tumors. Unfortunately, there
are no sufficient data about the efficacy of this treatment and
further studies are required.
Chemotherapy
In 1988 Averbuch reported for the first time that combination
of cyclophosphamide, vincristine and dacarbazine (CVD) was
effective against malignant pheochromocytoma [1]. The relatively
large series including 14 patients showed favorable biochemical
and tumor responses (79% and 57%, respectively)
with mean duration of 21 months and minimal side-effects,
mainly bone marrow toxicity and hypotension [1, 2]. Tada et
al described 3 patients with multiple metastases treated with
CVD chemotherapy and α-methyl-p-tyrosine. They showed
that this combination was safe and improved the clinical course
[26]. Sisson at al combined 131 I-MIBG therapy with
CVD in 6 patients with good response in 2 cases [25]. Unfortunately,
the effect of chemotherapy was transient without
clear impact on long-term survival. However, occasional
long-term survivors were described [15]. Thus, it is indicated
only in cases of failure of other types of treatment. [1, 15, 19].
External radiotherapy
External radiotherapy is not very effective. It is used as a palliative
treatment of chronic pain in patients suffering from
bone metastases and symptoms of local tumor compression
[19, 23, 28]. However, there is no data about efficacy of radiation
therapy in pheochromocytoma.
Other methods
There are some data in the literature describing successful
embolization and radiofrequency ablation of pheochromocytoma
metastases [28].
77

78
Patients with persistent hypertension need chronic
pharmacotherapy based on α and β receptor antagonists. Calcium
channel blockers also manage an effective blood pressure
control without overshoot and orthostatic hypotension
[2-5, 12, 28]. In case of intolerance or resistance to α-blockers
α-methylparatyrosine can be used to decrease tumor catecholamine
production [2, 28]. There are also some clinical
experiences with unlabeled somatostatin analogues, but the
first results are not promising and no tumor regression or hormone
level reduction were observed [28].
Follow-up
Long term, lifetime follow-up is necessary in all patients with
pheochromocytoma because recurrent disease or distant metastases
can develop even more than 15–20 years after successful
resection of apparently benign primary tumor [6, 8,
10, 12, 13, 27, 28]. Follow-up should include blood pressure
References
1. Averbach SD, Steakley CS, Young RN,
et al (1988) Malignant pheochromocytoma:
effective treatment with a combination
of cyclophosphamide, vincristine
and dacarbazine. Ann Intern Med
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2. Bravo EL (1994) Evolving concepts in
the pathophysiology, diagnosis and treatment
of pheochromocytoma. Endocr
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3. Bravo EL (2004) Pheochromocytoma:
current perspectives in the pathogenesis,
diagnosis and management. Arq
Bras endocrinol Metab 48: 746–750
4. Bravo EL, Tagle R (2003) Pheochromocytoma:
state-of-the-art and future
prospects. Endocr Rev 24: 539–553
5. Ciftci AO, Tanyel FC, Senocak ME,
Buyukpamukcu N (2001) Pheochromocytoma
in children. J Pediatr Surg 36:
447–452
6. Eisenhofer G, Bornstein SR, Brouwers
FM, et al (2004) Malignant pheochromocytoma:
current status and initiatives
for future progress. Endocrine-Related
Cancer 11: 423–436
7. European Association of Nuclear Medicine
(2003) Guidelines for 131 I–metaiodobenzylguanidine
therapy. Eur J
Nucl Med 30: BP23–BP26
8. Fitzgerald PA, Goldfien A (2004) Adrenal
Medulla. In: Greenspan FS, Gardner
DG (Eds) Basic and clinical endocrinology.
Lange Medical Books/
McGraw-Hill New York, pp 439–477
9. Garaventa A, Gambini C, Villavecchia
G, et al (2003) Second malignancies in
children with neuroblastoma after combined
treatment with 131 I-metaiodobenzylguanidine.
Cancer 97: 1332–1338
10. Goldstein RE, O’Neill JA, Holcomb
GW, et al (1999) Clinical experience
over 48 years with pheochromocytoma.
Ann Surg 229: 755–766
11. Hoefnagel CA, Schornagel J, Valdes
Olmos RA (1991) 131 I-metaiodobenzylguanidine
therapy of malignant pheochromocytoma:
interference of medication.
J Nucl Biol Med 35: 308–312
12. Januszewicz W, Jarz¹b B, Januszewicz
A, Prejbisz A (2005) Malignant pheochromocytoma.
Arterial Hypertension
9: 132–140 (in Polish)
13. Januszewicz W, Prejbisz A, Januszewicz
A, Pêczkowska M (2002) guz
chromoch³onny-choroba o wielu obliczach.
Arterial Hypertension 6:
217–227 (in Polish)
14. Januszewicz W, Wocial B, Chodakowska
J et al. (1991) Z³oœliwy guz chromoch³onny.
Pol Tyg Lek 48: 664–667
(in Polish)
15. Kaltas G, Mukherjee JJ, Plowman PN,
Grossman AB (2001) The Role of chemotherapy
in the nonsurgical management
of malignant neuroendocrine tumours.
Clin Endocrinol 55: 575–587
16. Kaufman BH, Telander RL, van Heerden
JA, Zimmerman D, Sheps SG,
Dawson B (1983) Pheochromocytoma
an the pediatric age group: current status.
J Pediatr Surg 18: 879–884
measurement, biochemical tests and imaging studies. The first
evaluation of plasma metanephrine level or 24-hour urine collection
for fractionated catecholamines, metanephrines is recommended
approximately 2–6 weeks after the operation [8,
22]. The next examinations should be obtained quarterly during
the first year after surgery and further annually [6, 8]. Serum
chromogranin-A can be also a good tumor marker, but
only in patients with normal renal function [2, 4, 8]. Imaging
procedures including computed tomography (CT), magnetic
resonance imaging (MRI), 131 I or 123 I MIBG scintigraphy and
new modalities such as somatostatin receptor scintigraphy or
positron emission tomography (PET) should be performed on
the basis of biochemical test findings [22].
Concluding, successful treatment of malignant pheochromocytoma
still remains a challenge for scientists and clinicians.
Further investigations of genetic predispositions,
new molecular prognostic markers and new therapeutical approaches
are required.
17. Konings JE, Bruning PF, Abeling NG,
van Gennip AH, Hoefnagel CA (1990)
Diagnosis and treatment of malignant
pheochromocytoma with 131 I-metaiodobenzylguanidine:
a case report. Radiotherapy
and Oncology 17: 103–108
18. Lam MG, Lips CJ, Jager PL, et al
(2005) Repeated 131 I-metaiodobenzylguanidine
therapy in two patients with
malignant pheochromocytoma. J Clin
Endocrinol Metab 90: 5888–5895
19. Lio S, Napolitano G, Giuliani C, et al
(1991) A overview on the management
of malignant pheochromocytoma. J
Nucl Biol Med 35: 263–265
20. Mukherjee JJ, Kaltas GA, Islam N, et al
(2001) Treatment of metastatic carcinoids
tumours, pheochromocytoma, paraganglioma
and nedullary thyroid carcinoma
of the thyroid with 131 I-metaiodobenzylguanidine.
Clin Endocrinol 55:
47–60
21. Neumann DR, Bausch B, McWhinney
SR, et al (2002) Germ-line mutations in
nonsyndromic pheochromocytoma. N
Engl J Med 346: 1459–466
22. Pacak K, Linehan WM, Eisenhofer G,
Mc Clellan MW, Goldstein DS (2001)
Recent Advances in genetics, diagnosis,
localization and treatment of pheochromocytoma.
Ann Intern Med 134:
315–329
23. Pham TH, Moir Ch, Thompson GB, et
al (2006) Pheochromocytoma and paraganglioma
in children: a review of medical
surgical management at a tertiary
care center. Pediatrics 118: 1109–1117

24. Rose B, Matthay K, Price D, et al
(2003) High-dose 131 I-metaiodobenzylguanidine
therapy for 12 patient with
malignant pheochromocytoma. Cancer
98: 239–248
25. Sisson JC, Shapiro BM, Shulkin BL,
Urba S, Zempel S, Spaudling S (1999)
Treatment of malignant pheochromocytoma
with 131 I-metaiodobenzylguanidine
and chemotherapy. Am J Clin Oncol
22: 364–370
26. Tada K, Okuda Y, Yamashita K (1998)
Three cases of malignant phoechromocytoma
treated with cyclophosphamide,
vincristine and dacarbasine combination
chemotherapy and alpha-methyl-ρtyrosine
to control hypercatecholaminemia.
Horm Res 49: 295–297
79
27. Williams DT, Dann S, Wheeler MH
(2003) Pheochromocytoma – views on
current management. EJSO 29:
483–490
28. Yu J, Pacak K (2002) Management of
malignant pheochromocytoma. Endocrinologist
12: 291–299

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 81–87
© Copyright by Polish Paediatric Pathology Society Annals of
Paediatric differentiated thyroid cancer – differences in biology
and treatment
Daria Handkiewicz-Junak, Barbara Jarz¹b
Departments of Nuclear Medicine and Endocrine Oncology
Maria Sklodowska−Curie Memorial Cancer Center
and Institute of Oncology, Gliwice Branch
Gliwice, Poland
Abstract
Childhood cancer is not a one disease entity, but rather
a spectrum of different malignancies varying by type of histopathology,
site of disease origin, age and sex. In this age
group majority of malignancies is due to non epithelial malignant
neoplasms – leukemias and lymphomas, central nervous
system tumours, sarcomas or the embryonal cancers
such as neuroblastoma, retinoblastoma and Wilms’ tumours.
Carcinomas – malignant tumours of epithelial origin – are
very rare, particularly before the adolescents years. In contrast
to the adult population, where carcinomas comprise
overwhelming majority of malignancies, in children and
younger than 20 years they comprise only about 9% of all
cancers. Among them, differentiated thyroid cancer (DTC)
contributes to a distinctly higher morbidity than carcinoma
in any other localization (only malignant melanomas exhibit
the similar incidence) [2].
Incidence rates of thyroid carcinoma in children and
adolescent younger than 20 of age constitute about 2% of all
thyroid cancers. They are practically negligible in very
young children, although single cases, clinically evident already
in 4–6 months old children or even neonates were described
in the literature [23, 36, 53]. An average annual rate
Address for correspondence
Childhood differentiated thyroid cancer (DTC) is a rare disease in children accounting for only about 2%
of all thyroid cancers. It has some undeniable differences with adult DTC ranging from molecular biology
to clinical outcome: 1) higher susceptibility to carcinogenetic effect of ionizating radiation, 2) higher
prevalence of RET/PTC rearrangements, 3) larger primary tumor at diagnosis; 4) metastatic pattern and
features. Although recurrence rates tend to be higher in children, cause-specific mortality remains low. To
achieve it, a multidisciplinary approach comprises some combination of surgery, radioiodine (131I)
ablation, and thyroid hormone therapy applied at varying intensities is crucial.
Key words: differentiated thyroid cancer, children, biology
Daria Handkiewicz-Junak, M.D, Ph.D. Phone: +48 32 2789301
Wybrze¿e Armii Krajowej 15 Fax: +48-32-2789325
44-100 Gliwice E-mail: dhandkiewicz@io.gliwice.pl
Poland
Diagnostic
Paediatric
Pathology
of DTC per million increases sharply in the teenagers, being
at that time additionally influenced by sex. The age-specific
incidence rates for males and females begin to diverge at age
of 10 years and from age 13 the rates increase substantially
for females [2, 22] (Fig. 1).
Although thyroid cancer is a rare disease in children
any thyroid nodule should be viewed with suspicion and the
diagnostic approach should be more aggressive in children
than in adults. The mean incidence of thyroid carcinomas in
Fig. 1 Age distribution of differentiated thyroid cance

82
childhood thyroid nodules is about 25% ranging from 10 to
50% [37] and is higher than the average incidence of 5% to
15% in adult population with thyroid nodules [17].
Etiology and etiopathogenesis
There were two distinct peaks in the incidence of thyroid
cancer in children and young adults. The first rise, seen in the
middle of XX century, was due to previous irradiation of head,
neck and upper thorax as a form of therapy for childhood
conditions – acne, tinea capitis, enlarged tonsils and hemangiomas
[13, 31, 48]. The risk was significantly elevated
10–19 yr after exposure, peaking at 20–30 years, and decreasing
40 years after exposure [50]. After radiation ceased to
be used for the treatment of these conditions the peak of thyroid
carcinomas declined by half and ionizing radiation was
established as one of the most prominent risk factor for thyroid
cancer [6]. The second peak of thyroid cancer incidence
in children, observed in some East European countries
was related to an internal uptake of radioiodine 131I following
the Chernobyl disaster in 1986 [61]. The peak started
just 4–5 years after exposure, reaching its maximum in the
mid-1990s, and the disease developed mainly in children

anaplastic cancer, even if they show some degree of differentiation,
and are often found in microcarcinoma which do
not exhibit propensity to develop a clinically aggressive disease
[57].
Alternative way of MAPK signalling pathway is mutation
of BRAF gene. Among several mutations, a thymine
to adenine inversion in the nucleotide 1799 is the most common.
It results in a substitution of a valine with a glutamic
acid at residue 600 of the protein and lead to a constitutive
activation of BRAF kinase [10]. To date, multiple studies have
confirmed that BRAF mutation is the most common event
in sporadic adult papillary carcinomas and occurs in approximately
45% of all cases [63]. In contrast to adult papillary
carcinomas, paediatric tumours (both sporadic and radiation-induced)
have a low prevalence of BRAF mutations
(0–12%) [28, 38]. In addition to papillary carcinomas, BRAF
mutations are found in thyroid anaplastic and poorly differentiated
carcinomas, typically in those tumours that also
contain areas of well differentiated papillary carcinoma [39].
In those tumours, mutant BRAF is detectable in both well
differentiated and poorly differentiated or anaplastic tumour
areas, providing evidence that it occurs early in tumorigenesis
and predisposes to tumour dedifferentiation. Kamagai et
al. [27] observed only one case of BRAF mutation in 46 PTC
cases diagnosed under 15 years of age, and this was a rare
case of poorly differentiated cancer.
Apart from older age in several studies, the presence
of BRAF mutation has been found to correlate with more
frequent extrathyroidal extension, advanced tumour stage
at presentation, and tumour recurrence [8]. BRAF mutation
was found to be an independent predictor of tumour
recurrence, even in patients with stage I and II of the disease
[64]. Importantly, BRAF mutations have also been associated
with the decreased ability of tumors to trap I-131
and treatment failure of the recurrent disease [46, 64]. However,
the association between BRAF mutation and more
aggressive tumour behaviour has not been found in some
other studies [59].
It is a matter of future investigations to answer the question
whether differences in mutations as a function of age
accounts for the well documented but yet poorly understood
observation that age is a relevant prognostic indicator for patients
with papillary thyroid carcinoma.
Clinical presentation and outcome
It is generally believed that differentiated thyroid carcinoma,
recognised in patients younger than 45–50 years has a better
outcome than in older patients. Age at diagnosis is included
in majority of clinical scoring system of differentiated thyroid
carcinoma like AGES (patient age, histologic grade of
the tumour, tumour extent and size of primary tumour) or
MACIS (metastases, patient age, completeness of surgical
resection, local invasion and size of primary tumour) from
Mayo Clinic. In all classifications younger age at diagnosis
correlates with better prognosis. According to AJCC (American
Joint Committee on Caner) patients with distant meta-
83
stases but being younger than 45 years of age are considered
to be in stage II of disease while those older than 45 are in
stage IV.
Favourable outcome in younger patients is based on
low mortality rate. The highest specific overall survival is
observed in children and adolescents where it approaches
100%. However cancer deaths do occur in this age group [2].
Although mortality rates in children and adolescents
are much lower than in adults, in children disease is often more
advanced at presentation and there is a higher risk of disease
recurrence [6, 20, 24, 65]. Papillary microcarcinoma defined
as tumour less than 1–1,5 cm, is a rare diagnosis in children
and in most studies accounts for less than 3% of PTC
diagnosis [7,25] while in adults up to 36% of thyroid caner
are below 1 cm [51]. Zimmerman et al [65] described 9% of
tumors under 1 cm in children in comparison to 22% in adults.
The wide range of prevalence in published studies may represent
differing thoroughness of thyroid gland sectioning (e.g.
the number of sectioning levels), completeness of thyroidectomy,
the histological criteria for diagnosing papillary thyroid
cancer, population / geographic differences and possible
differs in intensity of thyroid screening. For example, Demidchik
et al. [11] in their recent study of radiation induced thyroid
cancer, reported that in 73% of children thyroid cancer
had less than 2 cm. Thus, it seems that the problem of children
with DTC diagnosed less than 1–2 cm in diameter is probably
important only in subgroups with extensive screening
due to radiation exposure but not in children with sporadic caner.
There is not only larger primary tumour, but also a higher
propensity for lymph node and distant metastases in
childhood DTC [20, 65]. An extremely high propensity for
lymph node metastases is exhibit by radiation induced PTC
(>80%). In sporadic PTC in children the incidence node metastases
is high and range from 40% to 60%. In our study,
where clinical outcome of DTC in children and adolescents
younger than 18 years of age was compared with young
adults, we showed that incidence of lymph node and distant
metastases was respectively two and four times higher in
children than in young adults (Table 1).
The rate of distant metastases at diagnosis of PTC
shows two peaks, first in childhood and the second in patients
older than 60 years [33]. In children, distant metastases
outside the lungs are very rare. The literature contains
only a few reports of bone lesions or other localization of distant
metastases [47]. Unlike adult lesions, pediatric pulmonary
DTC metastases are overwhelmingly miliary and seldom
nodular, and when detected radiographically, are almost
always functional [43, 62]. For example, among 95 Byelorussian
children with Chernobyl-induced DTC lung metastases,
92 (97%) had disseminated, and only 3 (3%), nodular
pulmonary radioiodine uptake [43]. This type of lung metastases
– disseminated involvement of miliary type – was also
typically seen by other groups [1, 12].
The high prevalence of functional metastases in pediatric
DTC may be related to differences in sodium iodide
symporter (NIS) expression. While NIS expression is redu-

84
Table 1
Clinical characteristic of children and young adults with DTC (lit)
ced in cancer cells, childhood tumors appear to have greater
and more frequently detectable expression than adult tumors
[42, 47]. In the absence of TSH stimulation, NIS expression
is undetectable in ~65% of papillary and ~56% of follicular
cancers in patients age < 20 years [42]. In contrast, NIS
expression is absent or reduced from normal in ~90% of
adult DTC, as assessed by reverse transcription polymerase
chain reaction [47]. Expression of other iodine transport-related
molecules, pendrin and apical iodide transporter
(AIT), also has been found to be reduced in pediatric
(Wiench, manuscript in preparation) as well as in adult DTC
[18, 29], but it is unclear if it expression is greater in childhood
DTC.
The greater NIS expression in pediatric than in adult
DTC implies greater differentiation and radioiodine responsiveness
in the former, which may be relevant to outcome.
In young patients, recurrence risk was increased in NIS-negative
versus NIS-positive tumors, even when TNM status and
treatment were similar [42]. The degree of NIS expression in
primary DTC lesions correlated with subsequent radioiodine
uptake in metastastases [4] and the clinical response of recurrences
[35].
The net major characteristic of pediatric versus adult
DTC is a generally higher recurrence rate. With 16.6 years’
follow-up, this rate approaches 40% in patients with PTC
diagnosed when

more efficient than use of lower activities of ~1,1 GBq. The
higher activities are also more efficient in detecting and treating
distant (mainly lung) metastases. As an alternative to
this fixed activity protocol, some centres give 3,7 MBq/kg of
body weight (range: 1,85–7,4 MBq/kg). Another alternative,
based on Reynolds’ calculations, is the use of diagrams adjusting
the adult activity to the age of the treated child, with
larger decreases in younger children [45]. According to this
system, a 15-year-old should receive about 5/6, a 10-yearold,
1/2, and a 5-year-old, 1/3 the adult activity.
References
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2. Bernstein L, Gurney J (1999) Carcinomas
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3. Bongarzone I, Vigneri P, Mariani L,
Collini P, Pilotti S, Pierotti MA (1998)
RET/NTRK1 rearrangements in thyroid
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family: correlation with clinicopathological
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4. Castro MR, Bergert ER, Goellner JR,
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Surgery 104: 1157–1166

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 89–92
© Copyright by Polish Paediatric Pathology Society Annals of
Blue Native Electrophoresis: an additional useful tool to study
deficiencies of mitochondrial respiratory chain complexes
Agnieszka Karkuciñska-Wiêckowska 1 , Katarzyna Czajka 2 , Micha³ Wasilewski 2 ,
Jolanta Sykut-Cegielska 3 , Maciej Pronicki 1 , Bo¿ena Cukrowska 1 , Ewa Pronicka 3 ,
Krzysztof Zab³ocki 2 , Jerzy Duszyñski 2 , Mariusz R. Wiêckowski 2
1 Department of Pathology
3 Division of Metabolic Diseases, Department of Pediatric
The Children's Memorial Health Institute
Warsaw, Poland
2 Department of Cellular Biochemistry
Nencki Institute of Experimental Biology, Polish Academy of Sciences
Warsaw, Poland
Introduction
Abstract
Blue Native polyacrylamide gel electrophoresis (BN-PAGE),
originally described by Schägger and von Jagow in 1991 [4],
is an elegant method to study protein complexes from mitochondrial
membranes. It is important that BN-PAGE enables
separation of membrane protein complexes from organelles
Address for correspondence
Several mitochondrial disorders are connected with the decreased activity of the respiratory chain which
leads to disturbance in cell metabolism and eventually to severe pathologic changes in the organism.
Localization and measurement of changes in the amount and activity of respiratory chain complexes can
provide important information of the disease etiology. Components of the mitochondrial respiratory chain,
located in the inner mitochondrial membrane and grouped in four complexes (additionally, mitochondrial
ATPase is called – complex V), are working together providing a pathway for redox processes. A wide
spectrum of methods enables studying the activity of respiratory chain complexes in tissues, cell cultures
and in isolated mitochondria. Under special experimental conditions the activity of each complex can be
measured separately using spectrophotometric methods. Acrylamide Blue Native Electrophoresis,
commonly used in molecular biology to study composition of protein complexes and protein-protein
interactions, can be successfully adapted for diagnosis of mitochondrial diseases connected with
abnormalities of the respiratory chain. BN-PAGE and in-gel activity assay can be successfully applied either
for detection of respiratory chain complex deficiency or for visualization deficiencies of individual complex
activities.
Key words: Blue Native Electrophoresis, mitochondria, respiratory chain complexes, mitochondrial disorders
Diagnostic
Paediatric
Pathology
or membrane fractions, keeping their properties and enzymatic
activities unchanged. BN-PAGE, supplemented by
other methods, e.g., in gel activity assay, spectrophotometric
measurement of the mitochondrial respiratory chain activity
and SDS-PAGE (as a second dimension), can be successfully
used in diagnosis of respiratory chain complex deficiencies.
Mariusz R. Wiêckowski 3 Pasteur Street, 02-093 Warsaw, Poland
Laboratory of Bioenergetics, Biomembranes and Metabolic Regulation tel. +48 22 589-23-72
Department of Cellular Biochemistry fax +48 22 822-53-42
Nencki Institute of Experimental Biology e-mail: m.wieckowski@nencki.gov.pl
Polish Academy of Sciences

90
Isolation of mitochondria and sample preparation
for BN-electrophoresis
Isolation of mitochondrial fraction from biopsies of skeletal
muscles and cultured human skin fibroblasts was made according
to the method previously described by Klement et al.
[3] and Van Coster et al. [2]. Briefly, skeletal muscles biopsies
from patients kept at –80 o C were defrost, cut into small
pieces and resuspended in homogenization buffer. 75 cm 2
flask of cultured patient’s fibroblast was trypsynized. Cells
were washed twice in PBS and resuspended in homogenization
buffer containing digitonin (8 mg/ml). Cells were incubated
on ice for 15 minutes and after were diluted twice with
homogenization buffer. Skeletal muscles and fibroblast were
homogenized in a glass Potter-Elvehjem homogenizer
with a motor-driven Teflon pestle on ice. The homogenate of
skeletal muscles was centrifuged at 1,500 × g for 5 min twice.
The final supernatant was collected and centrifuged at
10,000 × g for 10 min. The homogenate form patients fibroblast
was resuspended twice with homogenization buffer and
centrifuged at 10,000 × g for 10 min. At this step, final mitochondrial
pellets can be immediately use for sample preparation
or alternatively frozen in –80 o C for further use.
During the first step of sample preparation for
BN-PAGE, mitochondrial membranes are resuspended in the
aminocaproic acid buffer [1.5M 6-aminocaproic acid, 50 mM
Bis-Tris pH 7.0], what stabilizes protein complexes during solubilization
and electrophoresis. Then, protein complexes should
be solubilized in nonionic detergent. Digitonin is one of the
mildest. Dodecylmaltoside (a mild neutral detergent) is stronger
comparing to digitonin, whereas Triton X-100 shows intermediate
behavior. The choice of a detergent depends on the
stability of the protein complex of interest during solubilization.
Usually, to separate complexes of the mitochondrial respiratory
chain, n-dodecyl-β-D-maltoside is used for solubilization
of mitochondrial membranes. Samples are incubated on
ice for 20–30 minutes and then centrifuged 100 000 × g for
15 min to remove unsolubilized material. Protein concentration
in the supernatant was determined according to Bradford’s
method using Bio-Rad protein estimation kit [1].
The next step of sample preparation is an addition of
Coomassie Brilant Blue G250 dye to the solubilized material.
In BN electrophoresis the electrophoretic mobility of protein
complexes is determined by the size and shape of the complexes
and not by the negative charge of bound Coomassie
blue dye. Thus, proteins are not separated according to the
charge/mass ratio but according to size in acrylamide gradient
gels. Dye molecules bound to proteins impose a charge
shift that even basic proteins can migrate to the anode during
BN-PAGE [5].
Also, in a proper amount, Coomassie blue preserves
the structure of protein complexes. A detailed scheme of
sample preparation is presented in Figure 1. Samples prepared
in this way can be loaded on the gel directly or, alternatively,
can be stored for one month at –80 o C. As an example
of internal standard, sample prepared from bovine heart mitochodria
frozen at -80°C can be used.
Fig. 1 Sample preparation for BN electrophoresis
Gel preparation
To enhance separation of individual OXPHOS complexes,
a proper acrylamide gel gradient should be used. To study
usefulness of BN-PAGE in the diagnosis of mitochondrial
disorders, the acrylamide gel gradient of 5–12% and the size
of 1 mm/16 cm/20 cm was prepared. The ready to use “recipe”
for gel preparation, with calculated proportions of individual
compounds, is presented in Table 1. To avoid rapid
polymerization of the gel, it is necessary to pre-cool all solutions
before montage of the gradient mixer. The gradient
gel prepared in this way polymerizes at room temperature
about 20–30 minutes. Afterwards, stacking gel can be formed
and the gel is ready to use. Alternatively, it can be stored
for two days at 4 o C.
Table 1
Composition of the solutions for 5–12% acrylamide gel
formation
Stacking Separation Separation
(4%) (5%) (12%)
30% Acrylamide 0.9 ml 2.5 ml 6 ml
3 × Gel buffer 1.5 ml 4.997 ml 4.997 ml
92.5% Glycerol -------- 1.83 ml 3.25 ml
H2O 3.5 ml 5.673 ml 0.753 ml
TEMED 10 μl 15 μl 15 μl
10% APS 50 μl 40 μl 40 μl
Total 5.96 ml 15 ml 15 ml
Gel buffer (3x): 150 mM Bis-Tris, 1.5 M aminocaproic acid,
adjusted to pH 7.0 with HCl at 4 o C.

Electrophoresis
Generally, at the beginning samples are driven by the applied
voltage slowly until they have entered the stacking gel, typically
50 V for 30 min – 1 h. Afterwards the voltage is increased
to 300 V. When the front reaches 1/3 length of the gel,
cathode buffer B must be replaced by the colorless cathode
buffer (Cathode buffer A – without Coomassie). Electrophoresis
can be continued at 300 V until the blue dye front reaches
the end of the gel. It is important to run the gel in a cold
room or, alternatively, to use a central cooling core, which
prevents thermal band distortion and loss of complex activity
during electrophoresis. The optimal composition of electrophoresis
buffers is presented bellow:
Anode buffer: 50 mM Bis-Tris/HCl, pH 7.0, at 4 o C
Cathode buffer A: 50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7.0, at 4 o C
Cathode buffer B: cathode buffer A + 0.02% Coomassie Blue G-250
Typical picture of the BN-PAGE of rat heart mitochondria
(H) and mitochondria enriched membrane fraction
from human cultured fibroblasts (F), that we usually get is
presented in Figure 2. It is visible that in samples from cultured
skin fibroblasts, evaluation of the amounts of OXPHOS
complexes is more difficult than in rat heart mitochondria,
due to high background staining. In Figure 3 we show representative
BN-PAGE of rat liver and rat heart mitochondria.
Fig. 2 Typical picture of the Blue Native electrophoresis of bovine heart
mitochondria (H) – 30 μg and mitochondria from human skin fibroblasts (F)
– 30 μg (gel 5–12%)
Fig. 3 Different content of complex I in rat liver and heart mitochondria demonstrated by
BN-PAGE and spectrophotometric measurement of complex I activity
Comparison of protein complex profiles of these samples
shows that rat heart mitochondria contain higher amount of
complex I than mitochondria isolated from liver. This result
was confirmed by the measurement of the complex I activity
in mitochondria from both tissues using spectrophotometric
method. To observe similar activity of complex I it was
necessary to use 60 and 120 μg of rat heart and liver mitochondria
respectively. This result indicates that BN electrophoresis
is fully compatible with the spectrophotometric measurement
of OXPHOS activity. The use of densitometry can
considerably increase its sensitivity.
In-gel activity assay
During Blue Native electrophoresis OXPHOS complexes retain
their enzymatic activity [2]. To visualize activity of individual
respiratory chain complexes, the gel has to be incubated
at room temperature with the following solutions:
Complex I: In 3 mM Tris-HCl buffer (pH 7.4) dissolve: 1 mg
NADH and 5 mg nitrotetrazolium blue (NBT).
Complex II: To the 1.5 mM phosphate buffer (pH 7.4) add 5 mM
EDTA, 10 mM KCN, 0.2 mM phenazine methasulfate,
50 mM succinic acid and 5 mg nitrotetrazolium
blue (NBT).
Complex IV:In 9 ml 50 mM phosphate buffer (pH 7.4) dissolve
5 mg 3,3’-Diamidobenzidine tetrahydrochloride
(DAB), 200 μg catalase, 10 mg cytochrome c and
750 mg sucrose.
Complex V: In buffer containing: 35 mM Tris-HCl, 270 mM glycine,
14 mM MgSO 4 (pH 7.8) dissolve 0.2% Pb
(NO 3 ) 2 and 8 mM ATP.
When the color reaction is well visible, the gel can be
fixed in the water solution of 10% methanol and 1% acetic
acid. The gel so processed can be left in the fixation solution
for further analysis. In the case of complex V fixation should
be avoided because it causes disappearance of the white
band characteristic for the active form of complex V. Figure
4 presents a typical picture of the in-gel activity assay performed
for BN-PAGE of rat heart mitochondria. In the presence
of enzymatic active OXPHOS complexes characteristic
staining (color reaction) is observed. Results presented
Fig. 4 In gel activity assay after BN-PAGE of rat heart
mitochondria (30 μg) (gel 5–12%)
91

92
in Figure 5 indicate that combination of BN-PAGE and in-
-gel activity assay can be successfully applied for detection
of individual respiratory chain complex activity deficiencies
(in this case complex IV). As it is presented, only 10 μg of
mitochondria isolated from the skeletal muscle biopsy is sufficient
to measure activity of complex IV using in-gel assay.
Fig. 5 In gel activity assay for the evaluation of complex IV activity in
skeletal muscle mitochondria from healthy volunteer and patient with the
COX deficiency
Perspectives
A great advantage of the combined method of BN-PAGE and
in-gel activity assay is that relatively small quantities of tissue
are required for evaluating the amount and the activity
Bibliography
1. Bradford MM (1976) A rapid and sensitive
method for the quantitation of microgram
quantities of protein utilizing
the principle of protein-dye binding.
Anal. Biochemistry 72: 248–254
2. Van Coster R, Smet J, George E, De
Meirleir L, Seneca S, Van Hove J, Sebire
G, Verhelst H, De Bleecker J, Van
Vlem B, Verloo P and Leroy J (2001)
Blue native polyacrylamide gel electrophoresis:
a powerful tool in diagnosis of
oxidative phosphorylation defects. Pediatr
Res. 50: 658–65
of the mitochondrial respiratory chain complexes. Only
30 mg of heart muscle, 50 mg of skeletal muscle is needed
to isolate mitochondria and to perform BN electrophoresis
and in-gel activity assay [2]. Comparison of different methods
for isolation and separation of the respiratory chain
complexes shows that the resolution of BN-PAGE is higher
than other methods such as Superose 6 gel filtration or sucrose-gradient
ultracentrifugation.
Most of the mtDNA mutations are heteroplasmic, so
the corresponding catalytic activities of respiratory chain
complexes may vary significantly from cell to cell. Therefore,
to make a correct diagnosis of the deficiency of respiratory
chain complexes, other methods, as histochemical colorimetric
reactions (directly applied to the tissues and allowing
evaluation of the OXPHOS catalytic activity in
individual cells) and spectrophotometric technique (allowing
to measure OXPHOS activity in crude homogenates of the
tissue) should be used simultaneously with BN-PAGE.
Acknowledgment
3. Klement P, Nijtmans LGJ, Van den Bogert
C and Houstek J (1995) Analysis of
oxidative Phosphorylation complexes
in cultured human fibroblasts and aminocytes
by Blue-Native-Electrophoresis
using mitoplasts isolated with the help
of digitonin. Anal. Biochemistry 231:
218–224
Research was supported by Internal Project of The Children’s
Memorial Health Institute Nr 158/06 (principal investigator
– doc. B. Cukrowska), Internal Project of The
Children’s Memorial Health Institute Nr 161/06 (principal
investigator – doc. M. Pronicki) and Grant Nr PB
0890/PO5/2005/29 (principal investigator – prof. E. Pronicka).
4. Schägger H and von Jagow G (1991)
Blue native electrophoresis for isolation
of membrane protein complexes in enzymatically
active form. Anal. Biochem
199: 223–231
5. Wittig I, Braun H-P and Schägger H
(2006) Blue native PAGE. Nature Protocols
1: 418–428

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 93–96
© Copyright by Polish Paediatric Pathology Society Annals of
Comparison between the efficiency of hair follicle-
and epidermal-derived keratynocyte cell cultures
Tomasz Drewa 1 , Bartosz Nadolski 1 , Ilona Sir 2 , Artur Czaplewski 1 ,
Przemys³aw Ga³¹zka 3 , Andrzej I. Prokurat 3
1 Department of Tissue Engineering
3 Department of Pediatric Surgery
Nicolaus Copernicus University
Collegium Medicum in Bydgoszcz, Poland
2 Department of Pathology
Oncology Center
Bydgoszcz, Poland
Introduction
Abstract
Grafting of the autologous cultured human epithelium is
a standard treatment of the burned patients [1]. Hypertrophic
scarring and skin graft contracture are major causes of
morbidity after burn injuries. The application of cultured
epithelial autografts to burn wounds is known to reduce
scarring and contraction [6]. Clinical strategies to decrease
hypertrophic scar should include an attempt at early wound
closure with skin grafting or the application of cultured epithelial
autografts [5]. The use of cultured keratinocytes in
burned patients minimizes the areas of autologous skin harvesting
and reduces the amount of blood transfusions [14].
The crucial point of cellular therapy in the treatment of burned
patients is to obtain the maximal area of cultured kera-
Address for correspondence
Grafting of the autologous cultured human epithelium is a standard treatment of burned patients. The aim
of this study was to compare efficiency of hair follicle-derived keratinocyte culture and epidermal-derived
keratinocyte culture. Epidermal-derived and hair follicle-derived cultures were established and cultured
3 weeks without feeder layer at 37 o C and 5% CO 2 atmosphere. Cell viability, morphology and cytokeratin
expression was examined. Three (3) epidermal and 17 hair follicles keratinocyte cultures were established.
The cells in all cultures have epithelial-like morphology and were positive for cytokeratines. During the
3 weeks the 5 cm 2 of confluent monolayer was obtained from all follicles. Epidermal-derived keratinocytes
obtained from 3 rats covered 3 × 75 cm 2 flasks within 3 weeks. Our results show that hair follicle-derived
culture cannot serve as the only source to built autologous graft for burned patient.
Key words: burns treatment, epidermal-derived keratinocytes, hair-follicle keratinocytes
Diagnostic
Paediatric
Pathology
tinocytes monolayer for transplantation. The aim of this
study was to establish culture of keratinocytes from hair
follicle and compare it to the epidermal derived keratinocyte
culture.
Methods
Animals
Three Wistar male rats aging 6 months, which served as
a control group of another experiment were used in this
work. Specimens were obtained after the animals were sacrificed
using carbon dioxide overdose. The donor site had been
disinfected preoperatively with a 0.5% solution of chlorhexidine
in 70% alcohol. Hairless skin specimens of approximately
1 cm2 were harvested from 3 rats. Additionally the
Tomasz Drewa, M.D., PhD, FEBU Phone: +48525853737, Fax: +4852585374
Department of Tissue Engineering E-mail: tomaszdrewa@wp.pl
Chair of Medical Biology www.tisssue-engineering.webpark.pl
Karlowicza 24
85-092 Bydgoszcz, Poland

94
8 vibrissae follicles were dissected from each rat. Before dissection
from the skin each hair was cut 4 mm long.
Epidermal-derived primary rat keratinocyte culture
The epidermis was twice rinsed with a buffered saline
solution (PBS, Sigma, Germany) and then incubated overnight
in a 0.25% trypsin solution (Sigma, Germany) at 4oC. After the incubation, the basal layer of the epidermis, containing
the keratinocytes, was scraped off. The scraped-off keratinocytes
were placed in tubes and centrifuged at 500 g for
5 min. The supernatant was removed and the residue was suspended
in 5 ml of medium in T-flasks (Greiner, Germany).
The keratinocyte culture medium contained Dulbecco’s Modified
Eagle Medium (DMEM) and Ham’s F-12 at a 3:1 ratio,
supplemented with 10% FBS. The medium was supplemented
with insulin, transferin, triiodothyronine, hydrocortisone,
EGF and antibiotics: penicillin (50 U/ml) and
streptomycin (0.05 mg/ml) (Sigma, Germany) [15]. Cells
were cultured 3 weeks without feeder layer.
Hair follicle-derived primary rat keratinocyte culture
Dissected follicles were digested in enzymatic bath
(Dispase 12 mg/ml, Gibco, US) overnight at 4oC. Then the
follicles were digested 30 min in room temperature in 0.1%
Trypsin / 0.02% Edetate Disodium Dihydrate (Na2DDA) solution
(Sigma, Germany). One follicle was put in separate
well and pressed using the piece of glass. 2 ml of keratinocyte
medium contained DMEM and Ham’s F-12 at 3:1 ratio,
supplemented with 20% of foetal bovine serum and additives
described above was added to each well. Culture was conducted
3 weeks at 37oC and 5% CO2 atmosphere and proper humidity.
The presence of keratinocytes in the culture was confirmed
by an assessment of cell morphology and cytokeratin
expression with the use of wide-spectrum anti-cytokeratin antibodies
(Pancytokeratin MMF, Dako, Denmark). The dimensions
of monolayers were compared under inverted microscope
(Nikon Eclipse TS100, Japan) equipped with digital camera
(Nikon E5400, Japan). Cell viability was assessed using
trypan blue exclusion tests. Cell viability was presented as
a ratio of living cells to the total cell number in each culture.
Results
Three primary epidermal keratinocyte cell cultures were established
according to Rheinwald and Green method and 17
hair follicles were the source for primary cultures. The all
cultures were positive for cytokeratins. The epidermal-derived
keratinocytes started to grow on the 2nd day after isolation
(Fig. 1). The hair follicle-derived keratinocytes migrated
and attached to the flask surface after 14 days (Fig. 2).
The morphological features of keratinocytes monolayers growing
from hair follicle and epidermis were similar. The cells
in all cultures have epithelial-like morphology (Fig. 3 and
Fig. 4). During the observation time i.e. 21 days, the total size
of 5cm2 of confluent monolayer was obtained from all follicles.
Epidermal-derived keratinocytes obtained from 3 rats
covered 3 × 75 cm 2 flasks within 3 weeks. Cultures derived
Fig. 1 The epidermal derived keratinocytes started to grow on the 2 nd day
after isolation procedure. The clusters of dividing cells can be visible.
Inverted microscope, magnification 100×
Fig. 2 The hair follicle derived keratinocytes started to migrate and attached
to the growing surface on the 14th day after culture establishing. The black
area is the hair follicle from which cells migrate. Inverted microscope,
magnification 100×
Fig. 3 The epidermal derived keratinocytes covered the whole 75 cm 2
culture flask in the end of experiment i.e. on the 21st day. Inverted
microscope, magnification 100×

Fig. 4 The hair follicle derived keratinocytes started to form the confluent
monolayer after 3 weeks. The black area is the rat hair. Inverted microscope,
magnification 100×
from epidermis were characterized by small amount of the fibroblasts-like
cells within the period of the first week after
culture establishing. Those fibroblasts-like cells disappeared
within the 2nd week of culture. Cell viability in hair follicles
derived cultured was 85.1 + 7.0%. Epidermal-derived keratinocytes
were characterized by 80.7 + 11.2% viability.
Discussion
In recent years cultured human skin has been used as a source
of new skin to engraft onto damaged areas of burned patients,
representing one of the first therapeutic uses of stem
cells [2]. The hair follicle bulge area is an abundant, easily
accessible source of actively growing, pluripotent adult stem
cells. The hair-follicle stem cells provide an important accessible,
autologous source of adult stem cells for regenerative
medicine [7]. It was previously demonstrated that spreading
in vitro cultured human keratinocytes are autocrine-induced
phenomena [3, 20].
We have presented that keratinocytes growth is independent
of fibroblasts feeder layer. The wound healing processes
can be stimulated distinctly by Keratinocyte Growth
Factor [8]. Keratinocytes regulate the expression of keratinocyte
growth factor (KGF) in fibroblasts and decrease the connective
tissue activity during the end-stage of wound healing
[12, 21].
The lower region of the outer root sheath (ORS) of vibrissae
follicles of adult mice contains clonogenic keratinocytes
[13]. Human ORS cells can be induced to develop highly
differentiated epidermal equivalents [9]. Fusenig et al.,
observed that differentiation declining from epithelial cells
from epidermis (NEK) over hair follicle outer root sheath
(ORS) to hair matrix cells (HMC) [4]. In this work we have
shown that cell culture establishing from hair follicle and epidermis
have the similar morphology in vitro.
The success of keratinocytes grafting is depended on
the area covered with cultured epidermal cells. The total area
of confluent hair-derived monolayer was small (5cm2). The
surface of the epidermal-derived culture was several times
greater during the observation time. Thus we suggest, that the
hair follicle derived cultures are not sufficient for building
graft for burned patient. The hair follicle-derived culture could
be used as co-culture with epidermal cells to built cellular
grafts. The clinical relevance of stem cells lies primarily
in their therapeutic potential with reconstruction of epithelia
by reimplantation of autologous stem cells. However, the benefit-to-risk
ratio cannot yet be accurately estimated [18].
The strategy to use hair follicle-derived keratinocytes for
burned patient seems to be very attractive, but several problems
should be resolved. On of the most important is the time
necessary to obtain confluent layer. The culture of hair
follicle-derived cells started to grow after 2 weeks. Within
the same period of time epidermal keratinocytes was reaching
confluence. Till now there is no method to accelerate
hair follicle-derived cells growth and short the time of culture.
Burned patient requires immediate grafting.
The tissue-engineered, differentiated hair follicle-derived
equivalent has been used only in chronic patients treatment
i.e. vascular leg ulcers [19]. The management of chronic
patients does not require any hurry to get the confluent
culture. We thought that there is a possibility to enrich epidermal-derived
cultures with hair follicle-derived cells. This
improvement would have several advantages. Epithelial stem
cells have been implicated in wound healing [10]. The hair
follicle contains keratinocytes characterized by an extensive
proliferative capacity. Some of these cells are able to undergo
at least 130 doublings [16]. Keratinocyte graft with hair
follicle- and epidermal-derived cells would represent higher
proliferative potential. It is very important that follicular epithelium
is characterized with the expression of CD200 (OX-
2, transmembrane glycoprotein) which modulates tolerance
on graft [17]. The modulation of the inflammatory response
is crucial element of cellular therapy. The hair follicle-derived
keratinocytes are protected from ultraviolet radiation,
which is known mutagenic for human keratinocytes [11].
In conclusion, it seems that the hair follicle-derived
culture cannot serve as the only source to built autologous
graft for burned patient. The hair follicle-derived cells could
be used as a supplement for epidermal-derived autologous
graft in the future.
95

96
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of grafted autologous cultured human
epithelium. Br J Plast Surg 42: 35–42
2. Alonso L, Fuchs E (2003) Stem cells of
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U S A 100 Suppl 1: 11830–11835
3. Duinslaeger L, Verbeken G, Reper P et
al. (1996) Lyophilized keratinocyte cell
lysates contain multiple mitogenic activities
and stimulate closure of meshed
skin autograft-covered burn wounds
with efficiency similar to that of fresh
allergenic keratinocyte cultures. Plast
Reconstr Surg 98: 110–117
4. Fusenig NE, Limat A, Stark HJ, Breitkreutz
D (1994) Modulation of the differentiated
phenotype of keratinocytes
of the hair follicle and from epidermis.
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al (2006) Investigation of keratinocyte
regulation of collagen I synthesis by
dermal fibroblasts in a simple in vitro
model. Br J Dermatol 154: 401–410
7. Hoffman RM (2006) The pluripotency
of hair follicle stem cells. Cell Cycle 5:
232–233
8. Kopp J, Wang GY, Kulmburg P, et al
(2004) Accelerated wound healing by
in vivo application of keratinocytes
overexpressing KGF. Mol Ther 10:
86–96
9. Limat A, Hunziker T (2002) Use of epidermal
equivalents generated from follicular
outer root sheath cells in vitro
and for autologous grafting of chronic
wounds. Cells Tissues Organs 172:
79–85
10. Lyle S, Christofidou-Solomidou M, Liu
Y, et al (1998) The C8/144B monoclonal
antibody recognizes cytokeratin 15
and defines the location of human hair
follicle stem cells. J Cell Sci 111:
3179–3188
11. Nelson D, Gay RJ (1993) Effects of UV
irradiation on a living skin equivalent.
Photochem Photobiol 57: 830–837
12. Nowinski D, Hoijer P, Engstrand T, et
al (2002) Keratinocytes inhibit expression
of connective tissue growth factor
in fibroblasts in vitro by an interleukin-
1alpha-dependent mechanism. J Invest
Dermatol 119: 449–455
13. Oshima H, Rochat A, Kedzia C, Kobayashi
K, Barrandon Y (2001) Morphogenesis
and renewal of hair follicles
from adult multipotent stem cells. Cell
104: 233–245
14. Rab M, Koller R, Ruzicka M, Burda G,
et al (2005) Should dermal scald burns
in children be covered with autologous
skin grafts or with allergenic cultivated
keratinocytes? - “The Viennese concept”.
Burns 31: 578–586
15. Rheinwald JG, Green H (1975) Serial
cultivation of strains of human epidermal
keratyncytes: the formation of keratinizing
colonies from single cells.
Cell 6: 331
16. Rochat A, Kobayashi K, Barrandon Y
(1994) Location of stem cells of human
hair follicles by clonal analysis. Cell 76:
1063–1073
17. Rosenblum MD, Olasz EB, Yancey
KB, et al (2004) Expression of CD200
on epithelial cells of the murine hair
follicle: a role in tissue specific immune
tolerance? J Invest Dermatol 123:
880–887
18. Rzepka K, Schaarschmidt G, Nagler M,
Wohlrab J (2005) Epidermal stem cells.
J Dtsch Dermatol Ges 3: 962–973
19. Tausche AK, Skaria M, Bohlen L, et al
(2003) An autologous epidermal equivalent
tissue engineered from follicular
outer root sheath keratinocytes is as effective
as split-thickness skin autograft
in recalcitrant vascular leg ulcers. Wound
Repair Regen 11: 248–252
20. Tenchini ML, Morra F, Soranzo C,
Malcovati M (1995) Effects of keratinocyte-secreted
soluble factors on spreading,
number of dendrites and cell-cell
contacts of human epidermal melanocytes
and dermal fibroblasts: a quantitative
analysis. Epithelial Cell Biol 4:
143–155
21. Witte RP, Kao WJ (2005) Keratinocyte-fibroblast
paracrine interaction: the
effects of substrate and culture condition.
Biomaterials 26: 3673–3682

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 97–102
© Copyright by Polish Paediatric Pathology Society Annals of
Histopathologic features of Wilms’ tumor
in the light of the revised SIOP-01 classification –
one centre retrospective analysis of 44 cases
Jerzy Niedzielski 1 , Rafa³ Becht 1 , Katarzyna Taran 2
1 Department of Pediatric Surgery and Oncology
2 Department of Pathology
University School of Medicine,
Lodz, Poland
Introduction
Abstract
Wilms’ tumor (nephroblastoma) (WT) represents 8–10% of
the malignances that occur in the pediatric age group and is
the most frequent of common solid tumors in children
(except for CNS tumors) [5, 11]. The peak in the incidence
is observed between 1 and 5 years of age, and the tumor rarely
occurs in adolescents and adults [11]. Associated congenital
anomalies are found in approximately 10–15% of
children with WT, and the most frequent are urogenital defects
[9]. WT is thought to be composed of primitive metanephric
blastema. The classic nephroblastoma is made up of
varying proportions of three cell types – blastemal, stromal
Address for correspondence
Authors reviewed records of 44 children with Wilms’ tumor (WT), aged one month to 11 years, treated
between 1993 and 2001 (9 years). All patients underwent multimodal therapy according to the SIOP
protocols. 9 patients had favourable, 30 standard, 4 unfavourable, and one patient unclassified histologic
type of WT (SIOP-93). 36 children were in clinical stage I and II (81.8%) and majority had standard tumor
histology (68.1%). A blastemal subtype of WT was found in most of the patients with standard histology
(60%), while an anaplastic WT dominated within the unfavourable histology group (75%). Complications
occurred in 10 children (22.7%); 4 of them died (9.1%). Event-free survival (EFS) was 86.4%, overall
survival (OS) 90.9%. Follow up period ranged from 4 to 12 yrs. (mean 8.7). Simulated analysis (SIOP-01)
revealed that 5 patients (11.4%) with favourable histology moved to the intermediate risk type, while 18
(40.9%) with standard histology moved to the high risk type. The blastemal subtype became the most
common in high risk tumors (81.8%) and together with the anaplastic subtype was responsible for mortality
in this group (3/22 – 13.6%). A poorly differentiated epithelial subtype was the most frequent (41.2%) and
the only feature with decreased OS in intermediate risk tumors. In authors’ opinion, it should not be
considered as an intermediate risk histological feature of WT.
Key words: anaplastic and blastemal subtype, clinical pathological correlation, poorly differentiated
epithelial subtype, subtype, prognosis, Wilms’ tumor
Prof. Jerzy Niedzielski MD, PhD Phone: (4842) 6177711
Department of Pediatric Surgery and Urology Fax: (4842) 6177705
36/50 Sporna str. jniedzielski@usk4.umed.lodz.pl
91-738 Lodz, Poland
Diagnostic
Paediatric
Pathology
and epithelial, but they are not all present in every case. The
blastemal cells usually show distinctive patterns of rosettes,
tubules and pseudonodules. The stromal structure may contain
mesenchymal component with metaplasia to striated muscles,
cartilage and fat tissue [5, 9].
The prognosis of patients with WT is related to the tumor
clinical stage, histological features of the tumor and treatment
protocols. The overall survival rate in children with
WT is approximately 90% regardless the clinical stage [2, 4,
5, 8, 10, 11].
The aim of this study was to evaluate the course of disease
and results of therapy in patients with WT treated in
one centre in relation to histological tumor features according

98
to SIOP (International Society of Pediatric Oncology) classification
of 1993 and verified by means of classification
SIOP-2001 [1, 9, 10, 11].
Material and methods
Authors reviewed records of 44 children with WT treated at
the Department of Pediatric Surgery and Oncology of the
University School of Medicine in Lodz between 1993 and
2001. Follow up period ranged from 4 to 12 yrs. (mean 8.7).
There were 23 girls (52%) and 21 boys (48%), aged one
month to 11 years (mean age 5 years). All patients underwent
multimodal therapy according to the SIOP protocols. Treatment
was initiated with neoadjuvant chemotherapy (ACTD
– dactinomycin, VCR – vincristine) without histologic verification,
diagnosis being based on clinical presentation and
results of imaging studies. Surgical removal of the tumor
with involved kidney was performed after 4-week-long (35
patients) or 6-week-long (5 children) initial chemotherapy.
Restaging was performed and adjuvant chemotherapy was
administered after histologic diagnosis of the tumor was established
(ACTD – dactinomycin, VCR – vincristine, EPI –
epirubicin, IFO – ifosfamide, VP-16 – etoposide, CBDCA –
carboplatin). Radiation therapy was applied additionally in
10 children with locoregional progression of the neoplasm.
In 4 infants under 6 months of age the treatment began with
primary surgical removal of the tumor without neoadjuvant
chemotherapy.
The assessment of the clinical course of the disease included:
presence of distant metastases (M) detected at the diagnosis
or during therapy, local recurrence of tumor confirmed
by means of imaging studies, progression of the disease and
deaths. The overall survival rate was calculated in the entire
group of children with WT as well as in consecutive subgroups
related to the clinical stage and tumor histologic features.
The removed tumors were assessed histologically at
the Department of Pathomorphology of the Institute of Pediatrics
in Lodz according to the SIOP-1993 working classification
of renal tumors of childhood. Results were verified
at the Department of Pathology of the Institute of Mother and
Child in Warsaw. Patients were grouped in four pathological
categories:
Table 1
Group 1 – tumors with „favourable” histology (low grade
malignancy),
Group 2 – tumors with „standard” histology (medium grade
malignancy),
Group 3 – tumors with „unfavourable” histology (high grade
malignancy),
Group 4 – tumors of unclassified histology due to extensive
necrosis.
For the purpose of this work the histopathologic diagnoses
were reclassified according to the revised SIOP-2001
working classification of renal tumors of childhood to create
a simulated analysis of a clinical course. Patients were divided
in three groups: low, intermediate and high risk tumors.
The clinical staging of tumors was done following the
National Wilms’ Tumor Study Group (NWTSG) in the SIOP
modification [2, 5, 9]. The obtained results were subjected to
a statistical analysis using chi2 test and Yule ratio (Q).
Results
Sex, age and number of patients regarding histological types of Wilms’
Of 44 patients with Wilms’ tumor, favourable histology was
found in 9 children (20,5%), standard histology in 30 instances
(68,2%), unfavourable histology in 4 cases (9%) and in
one patient (2,3%) histological type of tumor could not be determined
due to its extensive necrosis, the effect of neoadjuvant
chemotherapy (SIOP-93). There was no predominance
of any of sexes observed in any of four histological groups.
The average age was comparable in patients with standard
and unfavourable tumor types. Children with favourable histological
features were significantly younger (p < 0,001)
(Table 1). Thirty six (36) out of 44 children were in clinical
stage I and II (81,8%) and the majority were classified as having
standard histological type of tumor (30/44 – 68,1%). Nine
patients with favourable tumor histology were in clinical
stage I (66,7%) and II (33,3%). Patients with standard histology
were distributed in four clinical stage groups, although
most of them were in stage I and II (23/30 – 76,7%). All
4 children with unfavourable histological features were classified
as stage II only, and the patient with uncategorized histological
type of WT was in stage IV (Table 2). Survival
analysis was performed in consecutive clinical stages in or-
Histological features Number of patients Sex Age Mean age
of Wilms’ tumor (%)
(SIOP-93) M F
I favourable 9 (20,5%) 4 5 1 mo – 4 yrs 2,2 yrs
II standard 30 (68,2%) 15 15 1 mo – 8 yrs 3,4 yrs
III unfavourable 4 (9,0%) 2 2 1 – 6 yrs 3,5 yrs
IV unclassified 1 (2,3%) 0 1 11 yrs –
TOTAL 44 (100%) 21 23 1 mo – 11 yrs 5 yrs

Table 2
Number of patients and survival rate in respective clinical stages of Wilms’ tumor (NWTSG) regarding histopathological
classification (SIOP-93)
Histological types of Clinical stage No of patients Survival rate
Wilms’ tumor (SIOP-93) (NWTS) (%) (%)
Favourable I
II
6
3
9 (20,5%)
6 (100%)
3 (100%)
9 (100%)
Standard I 16 14 (87.5%)
II
III
7
3
30 (68,1%)
7 (100%)
2 (66,7%)
IV 4 4 (100%)
Unfavourable II 4 4 (9,1%) 3 (75%)
Unclassified IV 1 1 (2,3%) 1 (100%)
TOTAL 44 (100%) 40 (90,9%)
der to examine possible correlation between the course of disease
and clinical stage of tumor determined at the beginning
of disease following NWTS (Table 2). But no such correlation
was found in the examined group. Complications of neoplastic
disease were observed in ten children (22,7%). Metastatic
disease was present in 4 cases (9,1%); in two patients
at the diagnosis and in two during therapy with lung (3/4 –
75%) and liver (1/4 – 25%) involvement. There were 6 relapses
(13,6%) and four deaths (9,1%). The highest diversity of
the course of disease was observed in children with a standard
histological type of tumor. In this group of patients there
were 3 cases of metastatic disease, five relapses and three
deaths. Out of 4 patients with unfavourable histological type
one child died after recurrence and progression of disease
(Table 3). The remaining 38 patients (86,4%) completed the
Table 3
27 (90%)
therapy and are free of disease with follow up period ranging
from 3 to 11 years (mean 7,7 years). Event-free survival
(EFS) was 86.4% as compared to overall survival (OS) of
90,9%. Survival rate of 100% was observed in patients with
favourable and unclassified histological type of tumor. In the
group of standard histology, 90% of children were cured
compared to 75% survivors in the group of unfavourable histology
(Table 2). Comparison of OS between SIOP-93 and
simulated SIOP-01 analysis revealed increase in strategic
groups of intermediate and high risk tumors (Tables 2 and 4).
With reference to introduction of the revised SIOP-
2001 working classification of renal tumors of childhood, the
authors performed „simulated” analysis of the clinical course
of disease and distribution of patients in consecutive clinical
stages. Simulation revealed that 5 patients (11,4%) with
Complications of Wilms’ tumor regarding histopathological classifications of SIOP-93 and SIOP-01
Complications SIOP-93 SIOP-01
Favourable Standard Unfavourable Unclassified Low risk Intermediate
risk
High risk
Metastases
(n=4)
None 3 (3bl) None 1 1 (cn) None 3 (3bl)
Recurrences
(n=6)
None 5 (3bl, 1da,1pde) 1 (bl) None None 1 (pde) 5 (4bl, 1da)
Deaths
(n=4)
None 3 (2bl, 1pde) 1 (da) None None 1 (pde) 3 (2bl, 1da)
TOTAL – 11 2 1 1 2 11
Legends: bl – blastemal subtype, cn – complete necrosis, da – diffuse anaplasia, pde – poorly differentiated epithelial subtype
(epithelial acc. SIOP-01)
99

100
Table 4
Number of patients and survival rate in respective clinical stages of Wilms’ tumor (NWTSG) simulated on the basis of the
revised SIOP-01 classification
Histological types of Clinical stage Number of patients Survival rate
Wilms’ tumor (SIOP-01) (NWTS) (%) (%)
Low risk tumors I 2 2 (100%)
II 2 5 (11,4%) 2 (100%) 5 (100%)
IV 1 1 (100%)
Intermediate risk tumors I 12 11 (91.7%)
II 4 17 (38,6%) 4 (100%) 16 (94.1%)
IV 1 1 (100%)
High risk tumors I 8 7 (87,5%)
II
III
8
3
22 (50%)
7 (87,5%)
2 (66,7%)
19 (86.4%)
IV 3 3 (100%)
TOTAL 44 (100%) 40 (90,9%)
favourable tumor histology moved to the intermediate risk
type, while 18 children (40,9%) with standard histology moved
to the high risk type, increasing the total number of children
in this group to 22 (50%) (Table 4). All but two patients
with complications of neoplastic disease were classified as
having high risk tumors in the simulated analysis based on
the revised SIOP-01 classification. The only death in the group
of intermediate risk tumors was the patient with poorly
differentiated epithelial feature of WT (Table 3).
Table 5
Analyzing the possible correlation between the stage
of disease and histological features of tumor according to
SIOP-93 classification, patients were divided into groups depending
on histopathological diagnosis. The majority of cases
with favourable histology were highly differentiated epithelial
and foetal-cystic subtypes. Most of the patients with
standard tumor histology had a blastemal subtype (18/30 –
60%), while within the unfavourable histology group an anaplastic
subtype of tumor dominated (3/4 – 75%) (Table 5).
Complications of Wilms’ tumor regarding histopathological classifications of SIOP-93 and SIOP-01
Histological Histological features of tumor Histological features of tumor
subtypes (SIOP-93) (SIOP-01)
of Wilms’ Favourable Standard Unfavourable Unclassified Low risk Intermediate High risk
tumor risk
Foetal-cystic 3 (100) 3 (100)
Fibrocystic 1 (100) 1 (100)
Epithelial (highly
differentiated)
4 (100) 4 (100)
Mesoblastic 1 (100) 1 (100)
Epithelial (poorly
differentiated)
7 (85,7) 7 (85,7)
Blastemal 18 (88,9) 18 (88,9)
Stromal 5 (100) 5 (100)
Diffuse anaplasia 3 (66,7) 3 (66,7)
Sarcomatous 1 (100) 1 (100)
Completely necrotic 1 (100) 1 (100)
TOTAL (n=44)
( ) OS in %.
9 30 4 1 5 17 22

Simulated SIOP-01 analysis showed that blastemal subtype
became the most common feature of high risk histology
(18/22 – 81,8%) and together with the anaplastic subtype was
responsible for mortality in this group (3/22 – 13,6%). A poorly
differentiated epithelial subtype was the most frequent
(7/17 – 41,2%) and the only feature with decreased OS in the
group of intermediate risk tumors (Table 5). Survival rate of
children with WT ranged in the present study from 66,7% to
100% depending on histological subtype of the tumor. Mortality
was associated with three histological subtypes: diffuse
anaplasia, low differentiated epithelial and blastemal (Tables
5 and 6).
Table 6
Survival rate in relation to histologic subtype of Wilms’
tumor
Histological Number Deaths Survival
subtypes
of tumor
of patients rate
Diffuse anaplasia 3 1 66,7%
Poorly differentiated
epithelial
7 1 85,7%
Blastemal 18 2 88,9%
All others 16 - 100%
TOTAL 44 4 90,9%
Discussion
More than thirty years ago Currie at al. noticed the correlation
between histological type of the tumor and clinical course
of the disease [3]. Beckwith at al. were next, whose studies
on WT histological structure allowed for the first time
to separate two histological groups of favourable and unfavourable
correlation with the clinical course [2]. Subsequent
histopathological classifications supervised by SIOP and
NWTSG created a third group: tumors with standard histology
or tumors of intermediate malignancy [4, 6, 9, 10].
The retrospective analysis performed by the authors
in the group of 44 patients with WT treated in one centre between
1993 and 2001 revealed the 9% incidence of tumors
with unfavourable histological features (Table 2) which was
comparable with the SIOP data [10]. However, in the light
of the revised SIOP-01 classification these data appeared to
be considerably lower and misleading. The simulation
(SIOP-01) performed by authors demonstrated clearly that
half of all treated patients (22/44 – 50%) were classified as
high risk tumors which was confirmed by the clinical course
of disease (Table 4). Such a spectacular and significant
(p < 0,0001) increase in number of patients of this group can
101
be explained by reclassification and moving the children
with blastemal tumor feature from the standard (SIOP-93) to
the high risk group (SIOP-01) (Table 5). The observation by
Reinhard et al. of a 90% incidence of intermediate risk and
7% only of high risk tumors differed markedly from our findings
[8].
The statistical dependence between patient’s age and
sex and histological type of tumor was not demonstrated. However,
the mean age of children with tumors of favourable
histology (2,2 years) was lower than mean age of patients
with standard and unfavourable types of tumor (respectively:
3,4 and 3,5 years). The low clinical stages (I and II) were
observed in tumors with favourable histology (low risk tumors
according to SIOP-01) while the high ones (III and IV)
in the standard type tumors (high risk tumors according to
SIOP-01). However, the clinical course of disease was not
statistically dependent on the tumor stage in the analyzed
group of patients.
In performing the study the authors examined the incidence
of complications of neoplastic disease (metastases,
recurrences, disease progression and mortality rate) as well as
the survival rate in relation to histological diagnosis. It appeared
that the highest incidence of metastases (3/30 – 10%), relapses
(5/30 – 16,7%) and fatal course (3/30 – 10%) that was
found in the group of standard histological features of tumor
according to SIOP-93 moved to the group of high risk tumors
after the SIOP-01 reclassification (Table 3) with corresponding
data of metastases – 13,6% (3/22), relapses – 22,7%
(5/22) and deaths – 13,6% (3/22). The OS after reclassification
increased with statistical significance from 90% to 94%
in the standard histology group (Q = 0,23) and from 75% to
86,4% in the high risk group (Q = 0,36) (Tables 2 and 4) and
the last one was higher than given in the literature [4, 7].
The overall survival rate of patients with WT presented
in this study differed in consecutive histological subtypes.
It was significantly lower (Q = 1,0) in patients with the
following subtypes of tumor independent of classification:
anaplastic (66,7%), poorly differentiated epithelial (85,7%)
and blastemal (88,9%). This clearly shows that not only anaplastic
and blastemal but also poorly differentiated epithelial
subtype of WT is associated with high malignancy and poor
prognosis in comparison with all other subtypes in which
a fatal course of disease was not observed. The authors have
paid particular attention to the poorly differentiated epithelial
feature of WT, which as the only one out of three discussed
histological subtypes was not included in the high risk tumor
group of the new revised SIOP-01 classification (the
common epithelial subtype of intermediate risk tumors).
This finding needs further investigation. If it proves
to be true, an adjustment of the classification should be considered.
In the authors’ opinion, because the survival rate of
patients with poorly differentiated epithelial subtype was
even lower than in children with blastemal subtype of tumor,
the former subtype should not be considered a standard histological
feature of WT.

102
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GPOH trial and study for the treatment
of patients with unilateral nonmetastatic
Wilms’ tumor. Klin Pediatr 216 (3):
132–140
9. Sawicz-Birkowska K, Rabczynski J
(1996) Histologiczna ocena nerczaków.
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Towarzystwa Pediatrów
Onkologów (SIOP). Podst Med
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10. Tournade MF, Com-Hougue C, Voute
PA et al. (1993) Results of the sixth International
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Wilms’ Tumor trial and study: a risk-adapted
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pp: 556–603 (in Polish)

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 103–107
© Copyright by Polish Paediatric Pathology Society Annals of
Outcome of wide liver resections in children
Adam Bysiek, Joanna Palka, Andrzej Zaj¹c, Sabina Kantorowicz,
Miros³aw Krysta, Bartosz Bogusz, Wojciech Górecki,
Anna Taczanowska-Niemczuk, Ma³gorzata Zamora
Department of Paediatric Surgery
Children's Hospital, Jagiellonian University
Krakow, Poland
Introduction
Abstract
Comparing to the adult population, partial hepatic resections
in children are not commonly performed. Primary hepatic
neoplasms, constituting less than two percent of all pediatric
tumors, are the major indication for resection [2, 4, 5, 14].
Approximately three quarters of all liver lesions are malignant.
Epithelial tumors, hepatoblastoma and hepatocarcinoma,
are the most common and account for 90% of hepatic
malignancies. One third of childhood liver tumors are benign.
They include vascular malformations, adenomas, focal
nodular hyperplasia, mesenchymal hamartomas and various
Address for correspondence
Hepatoblastoma, hepatocarcinoma and, much less often, focal nodular hyperplasia and mesenchymal
hamartoma are the major indications for liver resection. This paper presents the outcome for children treated
surgically because of liver tumors. Between January 1999 and July 2006, 13 partial hepatic resections were
performed in the Department of Paediatric Surgery Children's Hospital, Jagiellonian University. Indications
were hepatoblastoma in 8 patients (62%), focal nodular hyperplasia in 2 (15%), mesenchymal hamartoma
in 2 (15%), and metastatic nephroblastoma in 1 (8%). In 6 patients (46%) right hemihepatectomy was
performed, in 2 (15%) extended right hemihepatectomy, in 1 (8%) – right hemihepatectomy with lateral
part of the left lobe, in 3 patients (23%) left hemihepatectomy and in 1 (8%) posterio-lateral portion of the
right lobe was resected. There were no perioperative deaths. Eleven patients, including all of the patients
with benign lesions, continue to live and develop normally without signs of illness. One child with
hepatoblastoma died three months after surgery, there is one recurrence of hepatoblastoma. Six patients
after treatment of hepatoblastoma are disease free with median follow up of 56 months. Surgery is the
treatment of choice in resectable hepatic lesions. In the children with malignant tumors the chemotherapy,
followed by surgery gives a big chance of survival. Taking into account the low risk of complications of
liver resections and good outcomes associated therewith, we consider hepatic resection a better choice for
children with mesenchymal hamartoma and progressing FNH than the risk of malignant transformation and
progression possibly requiring hepatic transplantation in the future.
Key words: child, focal nodular hyperplasia, hepatoblastoma, liver resection, mesenchymal hamartoma
Adam Bysiek MD PhD Phone: +48-12-658-15-50
Department of Paediatric Surgery Fax: +48-12-658-13-25
Children’s Hospital, Jagiellonian University
265 Wielicka St.
PL-30-663 Kraków, Poland
Diagnostic
Paediatric
Pathology
types of cysts. Nowadays thanks to progress in understanding
of hepatic anatomy, achievements in pre and postoperative
multi drug chemotherapy, survival of children with hepatic
malignant tumors has improved [2, 4, 12, 14]. Hepatic resection
still remains a challenge for pediatric surgeons due to location
and risk of surgical complications. However, in specialized
centers good results of hepatic resections encourage
to operate not only malignant but also potentially dangerous
benign lesions such as mesenchymal hamartoma and selected
cases of focal nodular hyperplasia (FNH) [15]. This article
presents our experience with partial hepatic resection in
thirteen consecutive patients during seven years period.

104
Patients and methods
Between January 1999 and July 2006, thirteen partial hepatic
resections were performed in the Department of Paediatric
Surgery Children’s Hospital, Jagiellonian University.
Resections were performed on six girls (46%) and seven
boys (54%). Their ages ranged from 3 weeks to 15 years
(mean 4.2 years). The indication for surgery was hepatoblastoma
in eight cases (62%), focal nodular hyperplasia in two
cases (15%), mesenchymal hamartoma in two cases (15%),
and in one case hepatic metastase of nephroblastoma (8%).
Basic clinical data are described in Table 1.
Table 1
Clinical data of children with hepatic resections
ted in reduction of AFP levels. After completing the preoperative
chemotherapy the children were scheduled for surgery.
Based on a pretreatment CT examination two patients were
qualified as the PRETEXT III group, but after preoperative
chemotherapy, an intraoperative assessment of the extent
of the tumor allowed for single lobe resection. The upper abdominal
transverse incision was used in all of the cases. We
conducted five right hemihepatectomies, one left hemihepatectomy,
one right bisegmentectomy, and one right lobectomy
with left lateral bisegmentectomy. After surgery assessment
according to the Intergroup CCG/POG Staging System
showed that six children were in stage I, one in stage II
No Sex Age at diagnosis Diagnosis PRETEXT Operation Follow-up
(months) (months)
1 F 7 HBL III L lobectomy 31
2 F 5 days HBL II R lobectomy 84
3 F 49 HBL II R bisegmentectomy 54
4 M 25 HBL II R lobectomy 52
5 M 38 HBL II R lobectomy 91
6 M 32 HBL III R lobectomy and
L lateral bisegmentectomy
death
7 F 113 HBL II R lobectomy 25
8 F 69 HBL III R lobectomy recurrence
9 M 27 WT II L lobectomy 25
10 M 36 FNH III Extended R lobectomy 34
11 M 168 FNH II L lobectomy 44
12 F 0,4 MH II R lobectomy 109
13 M 21 MH III Extended R lobectomy 32
HBL – hepatoblastoma, WT – metastasis of Wilms tumor, FNH – focal nodular hyperplasia, MH – mesenchymal
hamartoma, L – left, R – right
Patients with hepatoblastoma
The age at the diagnosis among patients with hepatoblastoma
ranged between 3 days and 113 months (mean 41,6 months).
The open biopsy was electively used to establish diagnosis in
six patients. In one patient chemotherapy was started without
pathological assessment of a type of tumor relying on the
typical presentation and highly elevated concentration of serum
alfa-fetoprotein. One of the patients (5 days old newborn)
had urgent laparotomy because of oligovolemic shock after
massive bleeding to the tumor. During this procedure a biopsy
was taken. The extent of the tumor was assessed according
to the PRETEXT classification. Five children were in the 2nd
PRETEXT stage and three in the 3rd.
The preoperative chemotherapy was conducted according
to the SIOPEL-3 protocol. In all of the patients it resul-
and one in stage III of the disease. The SIOPEL- 3 protocol
was continued.
Patients with focal nodular hyperplasia (FNH)
One of the patients, three years old boy, was admitted to the
hospital because of respiratory infection. The initial diagnosis
of hepatic tumor was made incidentally during abdominal
ultrasound. The AFP level was not increased. The abdominal
CT revealed right hepatic lobe tumor (PRETEXT II).
The diagnosis of hamartoma was made upon the laparoscopic
biopsy. The child was observed for three years and was
asymptomatic. The control CT showed progression of the tumor
that extended to the medial portion of the left lobe (PRE-
TEXT III). We decided to remove the tumor and extended
right hepatectomy was performed.

In our second patient the initial ultrasound was performed
because of nonspecific abdominal pain. Abdominal
CT done afterwards showed left hepatic lobe tumor (PRE-
TEXT II), highly contrast enhancing. The initial diagnosis of
haemangioma was made. During one year observation period
the AFP level was low but enlargement of the tumor size was
observed. Angiography excluded haemangioma. The child
was scheduled for surgery and left hemihepatectomy was
performed. Pathological examination result was focal nodular
hyperplasia.
Patients with mesenchymal hamartoma
A twelve days old female newborn was admitted to our institution
with initial diagnosis of congenital hepatic tumor.
Abdominal CT revealed right hepatic lobe tumor (PRETEXT
II). AFP level was slightly increased. The patient was qualified
for primary resection of the tumor. When the child was
nineteen days old a right hemihepatecomy was performed.
Pathological diagnosis revealed mesenchymal hamartoma.
Another patient, ten years old boy, was brought to our
hospital after abdominal trauma. Abdominal ultrasound revealed
liver tumor. Giant hepatic tumor of I, VII, VIII and
partially V and VI segments was observed in contrast CT.
AFP level was normal. After 5 months of observation and no
signs of regression of the tumor we decided to resect the lesion.
Right extended hepatectomy was performed. Pathological
examination revealed mesenchymal hamartoma.
Patient with metastase of nephroblastoma
Two years old boy was admitted to our hospital because of
huge left kidney tumor. Abdominal and chest CT scans sho-
Table 2
Clinical data of children with hepatic resections (continued)
wed metastatic tumors in left liver lobe (PRETEXT II) and
in lungs. Preoperative chemotherapy for nephroblastoma was
given. Left kidney with tumor was removed and left hepatectomy
was performed. Chemotherapy resulted in regression
of lung lesions.
Results
105
In none of the patients the volume of intraoperative blood
transfusion exceeded 50 ml per 100 ml of circulating blood
(Table 2) In two children it was not needed. In five patients
no drains were left in abdominal cavity. In the rest of the children
(except patient No. 1 with right hepatic duct lesion) drains
were kept for 1–3 days.
There were no perioperative deaths. There was one
major perioperative complication. During surgery in patient
No. 1 incidental lesion of right hepatic duct was diagnosed.
The absorbable tissue adhesive was used to close the defect.
After surgery there was a prolonged bile drainage from the
peritoneal cavity. Conservative treatment resulted in recovery
and no further surgery was needed. Oral feeding was introduced
on days 1–3 in all patients except patient No. 1. Eleven
patients, including all of the patients with benign lesions,
are alive and develop normally without signs of illness. There
was one death in patient No. 6, with hepatoblastoma (PRE-
TEXT III, POG III) three months after surgery, because of
rapid progression of the illness. There was one recurrence of
hepatoblastoma (PRETEXT III, POG II) in patient No. 8, six
months after primary surgery. In this patient the primary tumor
was adjacent to the inferior vena cava. Second surgery
with nonanatomic resection of the lesion was performed. In
No. Weight Intraoperatively Percentage Peritonealdrainage Oral feeding Antibiotics
(kg) transfused of circulating (days) introduction post. op.
blood volume blood (days post.op.) (days)
(ml) (%)
1 7.8 0 0 8 20 42
2 3.8 0 0 3 4 8
3 19.2 150 9,7 0 3 4
4 12.4 440 41,7 1 2 7
5 12.6 230 21,5 1 2 7
6 16.7 440 32,9 3 4 2
7 26.6 660 30,7 0 4 10
8 19.5 220 14,1 2 3 5
9 13 220 20,1 0 3 8
10 19.7 240 15,2 0 1 6
11 53 540 14,5 3 3 5
12 2.3 100 48,3 1 3 11
13 12.5 440 41,3 0 3 5

106
those two children (patient No. 6 and No. 8) the regression
of AFP level after initial chemotherapy and surgery was less
than 90% (68% and 87% respectively) as opposed to the rest
of the patients where regression was over 90% (Table 3) Six
patients after treatment of hepatoblastoma are disease free
with median follow up of 56 months. The disease free survival
is showed in Table 1. The patient who underwent hepatic
resection because of nephroblastoma metastasis is in good
condition, with no signs of illness.
Table 3
Serum alfa fetoprotein level in patients with hepatoblastoma
No AFP levels (ng/ml) Regression rate
Preoperative Postoperative (%)
1 5 667 202 96
2 37 680 231 99
3 10 530 834 92
4 57 700 922 98
5 5 856 313 95
6 74 150 23 870 68
7 114 860 3 99
8 125 305 16 437 87
Discussion
Multi drug chemotherapy and surgical resection of the tumor
are well established standards in treatment of hepatoblastoma.
The treatment of hepatoblastoma is an example of the
great advances made in pediatric oncology over the past few
decades. This has been achieved by advances in surgical
techniques and the use of chemotherapy [4]. Five year survival
rates are higher than 70% [12, 14].
Focal nodular hyperplasia (FNH) is a relatively rare
benign liver tumor, often asymptomatic and discovered incidentally
[6, 11]. It is increasingly being diagnosed as a result
of the widespread use of ultrasound, computed tomography
in the evaluation of patients with non-specific abdominal
symptoms [7]. There is no agreement on the malignant potential
of FNH. The histopathological features of a fibrolamellar
variant of hepatocarcinoma (FL-HCC) suggest a direct
link between this tumor and FNH and some authors hypothesize
a direct evolution from FNH to FL-HCC [13, 17].
There were reports on cases of hepatocellular carcinoma arising
within FNH [9, 13]. The main problem in management
derives from the frequent difficulty in differentiating FNH
from adenoma and fibrolamellar hepatocellular cancer. It is
well known that FNH may regress spontaneously [3], but in
some cases there may be a progression of the lesion [8]. In
our series we encountered two cases of FNH with tendency
to progress. The decision to resect the tumors was made to
protect children from potential involvement of all liver tissue.
Fig. 1 Right lobe with tumor prepared to resection
Fig. 2 Right lobe removed
Fig. 3 Resected left lobe with tumor
Hepatic mesenchymal hamartoma is a hamartomatous
growth of mesenchymal tissue in the liver of uncertain
etiology, but cytogenetic studies have suggested that the tumor
may be a neoplasm rather than a hamartoma [16]. It is
a space occupying lesion that can potentially compress adjacent
organs resulting in various complications including death
[1, 15]. Although rare, malignant transformation to embryonal
sarcoma (malignant mesenchymoma) has been re-

ported [10]. In some cases a spontaneous regression can be
observed [16]. Because of the potential malignant transformation
and size enlargement most of the authors agree that
the treatment of choice should be surgical resection [15]. The
authors of this paper agree with that kind of therapeutical approach.
Taking into account a relatively low risk of compli-
References
1. Arfa MN, Gharbi L, Zaafrani MR, et al
(2003) Cystic mesenchymal hamartoma
of the liver report of a case and review
of the literature. Hepatogastroenterology
50 suppl. 2: ccxlix: ccli
2. Carceller A, Blanchard H, Champagne
J, St-Vil D, Bensoussan AL (2001) Surgical
resection and chemotherapy improve
survival rate for patients with hepatoblastoma.
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755–759
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A, Buscarini E, Buscarini L (1996) Natural
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of the liver: An ultrasound study. J
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OJ (1995) An 8-year experience of
hepatic resection: indications and outcome.
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Motola-Kuba D (2006) Focal nodular
hyperplasia and hepatic adenoma: a review.
Ann Hepatol 5 (3): 206–211
8. Okada t, Sasaki F, Kamiyama T, et al
(2006) Management and algorithm for
focal nodular hyperplasia of the liver in
children. Eur J Pediatr Surg 14 (4):
235–240
9. Petsas T, Tsamandas A, Tsota I, et al
(2006) A case of hepatocellular carcinoma
arising within large focal nodular
hyperplasia with review of the literature.
World J Gastroenterol 28; 12 (40):
6567–571
10. Rammanujam TM, Ramesh JC, Goh
DW, et al (1999) Malignant transformation
of mesenchymal hamartoma of the
liver: case report and the review of the
literature. J Pediatr Surg 34: 1684–1686
11. Reddy KR, Kilgerman S, Levi J, et al
(2001) Benign and solid tumors of the
liver: relationschip to sex, age, size of
tumor, and outcome. Am Surg 67:
173–178
12. Roebuck DJ, Perliongo G (2006) Hepatoblastoma:
an oncological review. Pediatr
Radiol 36 (3): 183–186
107
cations associated with liver resections as well as good outcomes
resulting therefrom, we consider hepatic resection
a better choice for children with progressing FNH and with
mesenchymal hamartoma than the risk of malignant transformation
and progression possibly requiring hepatic transplantation
in the future [8, 15].
13. Saul SH, Titelbaum DS., Ganslet TS, et
al (1987) The fibrolamellar variant of
hepatocellular carcinoma. Its associatin
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60: 3049–3055
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Pediatr Surg 15 (1): 17–24
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mesenchymal hamartoma: a short
review. Arch Pathol Lab Med 130 (10):
1567–1569
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1681–1690
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carcinoma of the liver: the malignant
counterpart of focal nodular hyperplasia
with oncocytic change. Am J Clin Pathol
81: 521–526

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 109–113
© Copyright by Polish Paediatric Pathology Society Annals of
The study on the pathogenesis of intestinal dismotility
and malabsorbtion in gastroschisis on experimental model
– chicken embryo
Hanna Bu³hak-Guz 1 , Regina Cybulska 2 , Ewa Czichos 3 , Tomasz Krawczyk 3 ,
Andrzej Kulig 3 , Andrzej Chilarski 1
1 Department of Pediatric Surgery and Urology
3 Departament of Clinical Pathomorphology
Polish Mothers's Health Institute
Lodz, Poland
2 Institute of Histology and Embriology of Agricultural Academy
Lublin, Poland
Introduction
Abstract
In gastroschisis (G) bowel loops protrude through a narrow,
paraumbilical defect in the abdominal wall of the fetus into
the amniotic cavity and float freely in the amniotic fluid
exposed to its irritating activity leading to aseptic perivisceritis.
Impairment of peristalsis of the bowels and food malab-
Address for correspondence
Gastroschisis (G) is a congenital defect of the abdominal wall characterized by translocation of intestinal
loops outside the abdominal cavity during fetal life through a defect usually located on the right side of the
umbilical cord. Translocated fragment of intestines in the extraembryonic cavity is exposed to direct contact
with the amniotic fluid (AF) and excrements contained in it (urine and meconium). The abnormalities of
intestinal function vary in intensity and the question arises whether dysfunction of the alimentary tract
results form morphological changes in the whole intestinal wall or only in the translocated loops. The
designed experiments are aimed to answer this question, and also to determine the character and extension
of changes in the abdominal wall. The study consisted of an experimental creation of G on chicken embryos
model and the subsequent comparison of the histopathological picture of the bowel wall of the
intraabdominal loops versus the exteriorized ones. The histopathological examination revealed lesions in
the eviscerated loops (exposed to AF activity) including the congestion of the intestinal villi, accumulation
of erythrocytes in the villus vessels, concentration of inflammatory small-round cells in the serosa. Our
results showed that: 1) the experimental studies on G showed the differences in the histopathological picture
of the eviscerated bowel loops as compared to the intraabdominal ones, 2) lesions in the intestinal
epithelium (the congestion of the villi) result from the bowel constriction and can be responsible for
malabsorption in G patients, 3) inflammatory lesions in the serosa are caused by the irritating influence of
the AF and can be responsible for postnatal peristalsis impairment.
Key words: chicken embryo, gastroschisis, intestinal damage
Hanna Bu³hak-Guz Phone: + 48 42 2712136
Department of Pediatric Surgery and Urology E-mail: hbulhak@wp.pl
Polish Mothers's Health Institute
Rzgowska St. 281/289
93-338 Lodz, Poland
Diagnostic
Paediatric
Pathology
sorption occurring in newborns treated for G are a serious clinical
problem and have an influence on the method, length
and costs of the treatment [17].
In relation to the occuring bowel malfunction of various
severity, the question arises whether the alimentary
tract disfunction results from the morphological changes in
the bowel wall in the eviscerated loops only or perhaps they

110
exist in the entire intestine and whether the changes of the AF
composition will protect the bowels floating in it against the
formation of these lesions.
The aim of the study
The aim of the study was to explain the pathogenesis of the
intestine malfunction in the course of G on the experimental
model (the chicken embryos) and to answer the question
whether the morphological changes in the bowel wall, which
may be the underlying cause of the consequent functional disturbances
of the alimentary tract, refer to eviscerated loops
only or the whole intestine. If this is the case what is the character
of these lesions?
Material and method
The experimental research was conducted in the Department
of Laboratory Animals at the Polish Mother's Health Institute
(PMHI) with the consent of The Local Ethics Committee
for Research on Animals.
The eggs with 9-day-old chicken embryos (Gallus domesticus)
obtained from the Poultry Hatching Farm in
Rzgów were incubated in a hatching chamber at the temperature
of 37,7 Celsius, 60% humidity and the tray rotation
every two hours. On the 14 th of embryonic life (which corresponds
with the second trimester in mammals), G was created
experimentally according to the accepted experimental
model under the surgical microscope guidance with the use
of microsurgical technique (Fig. 1) [18].
Following the closure of the egg shell with the sterile
dressing, the embryos were replaced to the hatching chamber
and further incubation took place till the 20 th day. For the
assessment of the dynamics of morphological changes occurring
in the bowels, the creation of G was performed on the
Fig. 1 The view of created gastroschisis in operating microscope (11 th day
of incubation)
subsequent days of embryonic development i.e. from 11 th to
14 th day.
The experiment was completed while at least 3 live
embryos with G from each experimental day were achieved.
The sacrificed embryos were measured and weighed
for the general assessment (Fig. 2). Following the macroscopic
evaluation of the abdominal wall and eviscerated bowels,
the whole removed intestine was divided into 2 parts:
intraabdominal loops and eviscerated loops – positioned
outside the abdominal cavity and then fixed in 4% buffered
formaline solution. The control group was constituted by
the bowels deriving from the embryos no manipulation was
performed on and the group of sham operations from the
corresponding developmental days, divided into 2 portions
either. The histological preparations (routine hematoxyline
and eosin staining) was performed at the Clinical Pathomorphology
Department of PMHI and the microscopic specimens
were evaluated in a light microscope independently
in 2 centres: the Clinical Pathomorphology Department of
PMHI and the Institute of Histology and Embryology of the
Agricultural Academy in Lublin. There were specimens deriving
from 34 embryos: 17 from the control group and 17
from the G group.
Fig. 2 The macroscopic view of gastroschisis in chicken (21 st day of
incubation)
Results
The specimens obtained from 34 embryos were assessed histopathologically:
17 from the G group and 17 from of the
control group (5 from 11 th day of incubation, 11 from 12 th ,
8 from 13 th and 10 from 14 th respectively) (Table 1).
In 2 cases no G was found (self-cure) and in 1 another
concomitant bowel obstruction was recognized (Fig. 3).
The histopathology of eviscerated bowel (G) exposed
to AF in 8 (57 %) cases revealed significant congestion of
the intestinal villi and the accumulation of erythrocytes in

Table 1
Type of procedure and number of operated chicken embryos
Type of manipulation Day of incubation Total
11 12 13 14
Control group
Sham operation
� No manipulation
� Isotonic salt solution
1 2 1 1 5
on amniotic membrane
� Isotonic salt solution
and cutting of amniotic
1 3 1 0 5
membrane 1 2 1 3 7
Total
Gastroschisis group
3 7 3 4 17
� Gastroschisis
� Gastroschisis and
2 2 4 6 14
intestinal atresia
� Spontaneous recovery
1 1
of gastroschisis 2 2
Total 2 4 5 6 17
Fig. 3 The macroscopic view of gastroschisis and intestinal atresia (21 st day
of incubation)
the villus vessels (Fig. 4). These alterations were not observed
in the bowel slides of the same embryos remaining inside
the abdominal cavity or in the control groups (Fig. 5).
No lesions were observed in the muscular membrane, whereas
in the serosa of the extraabdominal bowel inflammatory
small round-cells accumulations were present (Fig. 6).
In one case, gastroschisis was connected with intestinal
atresia. Abnormal histological picture was seen in both
parts of atretic bowel. There were small, flatten, pleomorphic
villi in proximal loop and quite normal built villi in distal loop
(Fig. 7).
A
B
Fig. 5 The control group – normal histological view of intestinal villi
111
Fig. 4 The microscopic view of eviscerated bowel loop – significant congestion
of the intestinal villi and the accumulation of erythrocytes in the villus
vessels: A) 11 th day of incubation, B) 14 th day of incubation
In other two cases, the spontaneous recovery of gastroschisis
was found with normal microscopic picture of intestinal
wall like in control group.

112
Fig. 6 The gastroschisis – inflammatory small round-cells in serous membrane
A
B
Fig. 7 The microscopic view of gastroschisis and intestinal atresia: A) The
proximal atretic loop – small, flatten, pleomorphic villi, B) the distal atretic
loop – normal villi
Discussion
Gastroschisis is a congenital anomaly of the abdominal wall
with small and large bowel evisceration outside the abdominal
cavity through a defect in the abdominal wall, usually located
right to the appropriately situated umbilical cord. Bowels
are not covered with the hernia sack. In many cases in
the 3 rd trimester of pregnancy aseptic perivisceritis develops
due to the irritating influence of the amniotic fluid. The wall
of the exteriorized gut is inflamed, thickened, with a fibrous
peel on its surface [14].
The etiology of the anomaly and its origin still remain
unclear.
Despite of significant decrease in the neonatal mortality
(from 30% in the 70’s of 20 th century to 4–10% currently),
the prognosis still remains serious connected with life-
-threatening developmental disturbances of the fetus (intrauterine
growth retardation – IUGR, prematurity, preterm
delivery, oligohydramnios, aseptic perivisceritis) and of the
newborn (from the growth retardation due to malabsorption
and peristalsis disorders to short bowel syndrome) [9, 16].
The origin of the lesions in the wall of eviscerated bowel
and its progression, both in experimental animals and in
humans with G depends on the two phenomena: 1) inflammatory
response of the bowel to the contact with AF and pro-
-inflammatory cytokines in it, 2) compression of the intestines
at the level of the defect in the abdominal wall and compression
– related ischemic lesions [11, 12].
In the case of a narrow defect in the abdominal wall,
compression of mesentery and its vessels may occur and subsequently
wall hypoxia and related consequences ranging
from the lumen constriction, to obstruction and even to the
selfamputation of the herniated loops. The constriction of the
defect and/or increasing oedema of the bowel wall can intensify
ischemic processes (vessel disaster theory), may cause
intrauterine fetal hypoxia and consequently lead to the intrauterine
growth retardation (IUGR), oligohydramnios and preterm
delivery [3].
The impact of AF on intestines as an irritating factor
contributing to inflammatory lesion of the bowel wall still remains
unclear. There are two hypothetical models explaining
the detrimental influence of AF: ‘urinary’ and ‘digestive’. In
the ‘urinary model’ it is thought that increasing in the course
of pregnancy osmolality of AF (predominantly comprising fetal
urine in the 3 rd trimester) damages the bowel wall of the fetus
[10, 19]. According to the ‘digestive’ model a deleterious
effect is mostly connected with digestive enzymes [2, 4, 6, 15].
Numerous experimental studies (on chicken embryos
and mammal fetuses) were conducted to investigate the influence
of urine ingredients and meconium digestive enzymes
on the bowel wall [6, 10, 15, 19]. Also AF composition
alterations (volume, density, chlorine and urea contents) dependent
on the gestational phase may be responsible for the
inflammatory lesions [8, 15]. Oligohydramnios accompanying
some of the cases of G and related increased concentration
of the aforementioned ingredients can be in a close relationship
with the pathological changes in the intestinal
wall. Currently, it is believed that the digestive enzymes of
the meconium are responsible for the inflammatory bowel lesions
(alkaline phophatase, enteric bicarbohydrates, trypsin).
The isolated presence of meconium in AF is not sufficient
since below a certain threshold level the induction of serous
enteritis does not occur. It may account for clinical observations
of varying severity of inflammatory lesions in the bowel
wall in neonates with the same defect – gastroschisis [4].
Previously conducted experimental studies focused
mostly in the factors triggering lesions in the bowels and the
type of lesions occurring in the bowel wall [1, 2, 7, 13, 15].

Presented results seems to prove that the ensuing lesions are
limited to eviscerated loops only. So it can be concluded that
the severity of motor and absorption disorders corresponds
with the capacity of eviscerated loops. The character of lesions
confirms both aforementioned pathomechanisms of bowel
damage (congestion of villi – compression and ischaemic
alterations, the accumulations of microround inflammatory
cells in the serosa – response to the irritating influence of AF).
Conclusions:
1. The experimental studies on G showed the differences in
the histopathological picture of the eviscerated bowel loops
as compared to the intraabdominal ones.
References
1. Aktug T, Erdag G, Kargi A, Akgur FM,
Tibboel D (1995) Amnio-allantoic fluid
exchange for the prevention of intestinal
damage in gastroschisis: an experimental
study on chick embryos. J Ped
Surg 30 (3): 384–387
2. Aktug T, Ucan B, Olguner M, Akgur
FM, Ozer E, Caliskan S, Onvural B
(1998) Amnio-allantoic fluid exchange
for the prevention of intestinal damage
in gastroschisis III: determination of the
waste products removed by exchange.
Eur J Pediatr Surg 8: 326–328
3. Albert A, Sancho MA, Julia V, Diaz F,
Bombi JA, Morales L (2001) Intestinal
damage in gastroschisis is independent
of the size of the abdominal defect. Pediatr
Surg Int 17: 116–119
4. Api A, Olunger M, Hakguder G, Ates
O, Ozer E, Akgur FM (2001) Intestinal
damage in gastroschisis correlates with
the concentration of intraamniotic meconium.
J Ped Surg 36 (12): 1811–1815
5. Bruner JP (2004) „Amnioexchange”
for gastroschisis. Ultrasound in Obstetricsss
and Gynecology, 24: 224
6. Correia-Pinto J, Tavares ML, Baptista
MJ, Henriques-Coelho T, Estevao-Costa
J, Flake AW, Leite-Moreira F
(2002) Meconium dependence of bowel
damage in gastroschisis. J Ped Surg 37
(1): 31–35
2. Lesions in the intestinal epithelium (the congestion of the
villi) result from the bowel constriction and can be responsible
for malabsorption in G patients.
3. Inflammatory lesions in the serosa are caused by the irritating
influence of the AF and can be responsible for postnatal
peristalsis impairment.
Acknowledgments
7. De Torre BL, Tovar A, Uriarte S, Aldazabal
P (1991) Transperitoneal exchanges
of water and solutes in the fetus
with gastroschisis. Experimental study
in the chick embryo. Eur J Pediatr Surg
1: 346–352
8. Kanmaz T, Yagmurlu A, Aktug T,
Gokcora H (2001) The effect of amnio-
-allantoic fluid pH on the intestines: an
experimental study in the chick embryo
gastroschisis model. J Ped Surg 36 (9):
1341–1345
9. Kimble RM, Blakelock R, Cass D
(1999) Vanishing gut in infants with gastroschisis.
Ped Surg Int 15: 483–485
10. Kluck P, Tibboel D, Van Der Kamp
AWM, Molenaar JC (1983) The effect
of fetal urine on the development of the
bowel in gastroschisis. J Ped Surg 18
(1): 47–50
11. Langer JC, Bell JG, Castillo RO, Crombleholme
TM, Longaker MT, Duncan
BW, Bradley SM, Finkbeiner WE, Verrier
ED, Harrison MR (1990) Etiology
of intestinal damage in gastroschisis, II.
Timing and reversibility of histological
changes, mucosal function, and contractility.
J Ped Surg 25 (11):
1122–1126
12. Langer JC, Longaker MT, Crombleholme
TM, Bond SJ, Finkbeiner WE, Rudolphc
A, Verrier ED, Harrison MR
(1989) Etiology of intestinal damage in
gastroschisis. I: Effect of amniotic fluid
exposure and bowel constriction in
a fetal lamb model. J Ped Surg 24 (10):
992–997
113
Authors are very grateful to the laboratory staff of the Department
of Laboratory Animals and the Department of Clinical
Pathomorphology, Polish Mothers’s Health Institute for
substantial support during this research.
13. Luton D, De Lagausie P, Guibourdenche
J, Pechmaur M, Sibony O, Aigrain
Y, Oury JF, Blot P (2000) Influence of
amnioinfusion in a model of in utero
created gastroschisis in the pregnant
ewe. Fetal Diagn Ther 15: 224–228
14. Luton D, Guibourdenche J, Vuillard E,
Bruner J, De Lagausie P (2003) Prenatal
management of gastroschisis: the
place of amnioexchange procedure.
Clin Perinatol 30: 551–572
15. Olguner M, Akgur FM, Api A, Ozer E,
Aktug T (2000) The effect of intraamniotic
human neonatal urine and meconium
on the intestines of the chick embryo
with gastroschisis. J Ped Surg 35
(3): 458–461
16. Stringel G, Filler RM (1979) Prognostic
factors in omphalocele and gastoschisis.
J Ped Surg 14 (5): 515–519
17. Sydorak RM, Nijagal A, Sbragia L,
Tsao K, Phibbs RH, Schmitt SK, Lee H,
Farmer DL, Harrison MR, Albanese CT
(2002) Gastroschsis: small hole, big
cost. J Ped Surg 37 (12): 1669–1672
18. Tibboel D, Molenaar JC, Van Nie CJ
(1979) New perspectives in fetal surgery:
the chicken embryo. J Ped Surg 14
(4): 438–440
19. Tibboel D, Raine P, Mcnee M, Azmy
A, Kluck P, Young D, Molenaar JC
(1986) Developmental aspects of gastroschisis.
J Ped Surg 21 (10): 865–869

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 115–119
© Copyright by Polish Paediatric Pathology Society Annals of
Humoral and cytokine responses induced by probiotic
Lactobacillus casei and paracasei strains in children
with atopic dermatitis
Bo¿ena Cukrowska 1 , Ilona Rosiak 1 , Aldona Ceregra 2 , Joanna Freszel 3 ,
Gra¿yna Zakrzewska 3 , El¿bieta Klewicka 4 , Ilona Motyl 4 , Zdzis³awa Libudzisz 4
1 Department of Pathology
2 Department of Paediatrics
The Children's Memorial Health Institute
Warsaw, Poland
3 Warsaw Agricultural University
Warsaw, Poland
4 Institute of Fermentation Technology and Microbiology
Technical University of Lodz
Lodz, Poland
Introduction
Abstract
Probiotics are live microorganisms that when ingested might
have a positive effect on disorders in which immunological
disturbances occur [7]. Experimental and clinical studies
have indicated that probiotics can significantly influ-
Address for correspondence
Probiotic bacteria have been shown to be useful in both prevention and treatment of atopic dermatitis (AD)
in children. Recently we indentified novel probiotic Lactobacillus casei and paracasei stains which
improved clinical syndromes of AD in children with cow’s milk (CM) allergy. The aim of the study was
to analyze the effect of those strains on production of secretory and circulating anti-Lactobacillus antibodies
as well as on modulation of serum cytokine profile. The study included 60 children with recognized AD
caused by CM allergy. Patients were randomized in a double-blind design to receive either placebo or the
mixture of Lactobacillus casei LOCK 0900, L. casei LOCK 0908 and L. paracasei LOCK 0919 for
3 months in daily dose 5×10 9 . Antibody and cytokine responses were measured using immunoenzymatic
methods before bacteria application, just after finishing bacteria in-take and 5 months later. We observed
an increase in antibody secretion mainly of IgA isotype in stools, but without statistical significance
(p=0,08). Cytokine profile analyses showed activation of proinflammatory IL-12 (p=0,06) and IL-18
(p=0,03) after 8-month lasting observation. We conclude that the improvement of clinical syndromes after
Lactobacillus bacteria in-take is dependent on modulation of cytokine balance, and in this process IL-18
seems to play the most important role.
Key words: allergy, antibody response, cytokines, IL-18, Lactobacillus, probiotics
Bo¿ena Cukrowska, MD, PhD fax: +48 22 8151975
Department of Pathology e-mail: b.cukrowska@czd.pl
The Children’s Memorial Health Institute
Aleja Dzieci Polskich 20
04-736 Warsaw, Poland
Diagnostic
Paediatric
Pathology
ence the immune responses of host in promoting the production
of secretory and circulating antibodies (Abs), and
altering the balance of proinflammatory (Th1)/proallergic
(Th2) responses and the cytokine production profile [2, 7,
15]. Recently, a role of probiotic bacteria, especially Lactobacillus
rhamnosus strain GG (LGG), both in prevention

116
and treatment of allergic diseases was demonstrated [8, 9,
13, 18, 19]. LGG administrated to pregnant women reduced
the incidence of allergy in their children after 2 and
4 years. Application of LGG to infants with atopic dermatitis
(AD) decreased the severity of the disease. This strain
induced an increase the proinflammatory cytokine response
in peripheral lymphocytes in infants with IgE-associated
AD and activated production of secretory IgA [11, 12, 16,
17]. It seems that antibodies, especially occurring on mucosa
could react with allergens protecting their translocation
through mucosal barrier. On the other hand improvement
of disease symptoms could be induced by activation
of Th1 cytokines which maintain Th1/Th2 balance in allergic
children [2, 7].
We identified novel probiotic Lactobacillus casei and
paracasei strains which given to children with cow’s milk
(CM) allergy demonstrating AD improved clinical syndromes
in IgE-allergic patients [1, 5]. The aim of this study was
to analyze the effect of probiotic Lactobacillus casei and paracasei
strains on production of secretory and circulating anti-Lactobacillus
Abs in sera and stools as well as on modulation
of cytokine profile in children with AD.
Patients and methods
Patients’ characteristics and study design
The study included 60 children aged 3 months – 18 months
(mean age 10 months) with atopic dermatitis caused by CM
allergy, who did not received antibiotics and probiotics for
at least 3 months before the study. CM allergy was proved
by challenge with CM formula. During the study children received
hydrolyzed milk formula or were breast fed. In case
of breast feeding women were on CMP-free diet. Patients
were randomized in a double-blind design to receive either
placebo or a mixture of three probiotic strains: Lactobacillus
casei LOCK 0900, L. casei LOCK 0908, L. paracasei LOCK
0919 for 3 months in daily dose 5×109 . Clinical improvement
was evaluated using Severity Scoring of Atopic Dermatitis
(SCORAD) before bacteria application (time 0), at the end
of their in-take (3 m) and 5 months later (8 m). At the same
time sera and faeces were collected for measurement of antibody
and cytokine levels. Finally, 44 children finished the
whole study and determination of Ab levels were done in 33
children. Faeces were diluted with PBS in concentration
1 g/ml, vortex and centrifuged. Fecal supernatants were collected
and frozen in – 20oC. The infants participated in the study with the informed
consent of their parents and the study was approved by
the Ethics Committee of the Children’s Memorial Health Institute.
Detection of specific anti-Lactobacillus Ab
The Ab response was estimated in the sera by enzyme linked
immunosorbent assay (ELISA) as previously described by
Cukrowska et al [4] using heat-inactivating mixture of probiotic
Lactobacillus bacteria as a antigen. Microplates were
coated with bacteria suspended in PBS at a concentration of
10 8 /ml. After overnight incubation bacteria were fixed to the
plates by a10-min incubation with 0,025% glutaraldehyde.
The plates were then washed with tap water and phosphate
saline buffer (PBS) pH 7,4, and blocked with 5% normal goat
serum (Sigma Chemical Co, St. Louis, MO, USA) for
30 min in room temperature. After washing samples duplicates
were applied. Serum samples were diluted in 1% bovine
serum albumin (BSA) /PBS in following dilutions: for
IgA Ab 1:50 and 1:200, for IgM 1:100 and 1:500, for IgG
1:200 and 1: 800. Fecal samples were applied without dilution.
Standard normal human adult serum was always added
in a five-fold dilution starting from 1:100 as a positive control,
1% of BSA being used as a negative control. After overnight
incubation at 4 o C and washing, polyclonal goat anti-human
IgA, IgG or IgM conjugated to peroxidase (Jacson Immnoresearch)
diluted 1:2000 in 1%BSA/PBS were added.
Finally, the plates were washed and 50 μl of solution containing
o-phenylenediamine dihydrochloride (Sigma Chemical
Co, St. Louis, MO, USA) was added. The reaction was stopped
with acid sulphuric and the plates were read on micro-
-ELISA reader at 450. The level of Ab activity was expressed
as arbitrary units (AU) calculating from the calibration
curve, in which standard normal human serum diluted 1:100
contained 100 AU.
Cytokine determination
In sera proinflammatory interleukin-18 (IL-18), IL-12, interferon-gamma
(IFN-gamma), regulatory transforming
growth factor –beta 1 (TGF-beta1) and proallergic IL-4, IL-
5 were measured. The cytokine level was determined as previously
described by Rosiak et al using R&D System Kits
[14]. Briefly, 50 μl of supernatants was added to a microtitter
well coated with the specific monoclonal antibody and
left for 24 hour in 4oC. After incubation the wells were washed
and 100 μl of detection antibody was added to each
well and the plates were incubated for 2 hours in room temperature.
Then the wells were washed again and 100 μl of
streptavidin was added for 20 minutes. Finally, the plates
were washed and 50 μl of solution containing o-phenylenediamine
dihydrochloride (Sigma Chemical Co, St. Louis,
MO, USA) was added. The reaction was stopped with acid
sulphuric and the plates were read on micro-ELISA reader
at 450. The amounts of cytokines were calculated from the
standard curve. The results were expressed in pg/mL as arithmetical
means.
Statistical analysis
The results were statistically analyzed using parametric t-test
and nonparametric Mann-Whitney test. P

in Ab levels mainly of IgA and IgM isotopes was detected
during 8-month lasting observation in patients receiving Lactobacillus
strains as well as in placebo group. Although Lactobacillus
bacteria in-take induced slightly higher amounts of
IgA anti-Lactobacillus Abs in sera as compared with sera of
children receiving placebo the statistical significance was not
achieved (Fig. 1). In stools IgA anti-Lactobacillus Abs dominated,
and only minimal amounts of IgM and IgG Abs were
found (Fig. 2). In contrast to circulating Abs which incre-
AU
AU
AU
3000
2500
2000
1500
1000
500
0
2500
2000
1500
1000
500
0
2000
1500
1000
500
0
IgA
0 m 3 m
Lactobacillus Placebo
IgM
0 m 3 m
Lactobacillus Placebo
IgG
0 m 3 m
Lactobacillus Placebo
Fig. 1 Specific anti-Lactobacillus Abs in sera of children receiving Lactobacillus
strains (black bars) and placebo (white bars)
Abs were measured by immunoenzymatic assays. There was no statistical
significance between Lactobacillus and placebo groups.
117
ased during 8-month observation in both groups, an increase
in Ab level in stools was observed only in children receiving
Lactobacillus strains. An increase was found only just after
finishing the bacteria in-take (3 m). After next 5 months Ab
amounts decreased to the level observed in placebo group.
An increase was found in all isotypes, but did not achieve the
statistical significance in patients supplemented with probiotics
in comparison with placebo group, although for IgA Ab
p=0,08.
Fig. 2 Specific anti-Lactobacillus Abs in stools of children receiving Lactobacillus
strains (black bars) and placebo (white bars)
Abs were measured by immunoenzymatic assays. *statistical significance
between children receiving Lactobacillus strains and placebo.

118
Cytokine level in sera
IL-4 and IL-5 was not detected in most (>90%) sera, and
IFN-gamma was found in 5 children with group receiving
probiotics and in 4 from control placebo group. IL-12, IL-18
and TGF-beta1 were detected in sera of all children. The statistical
significance between studied groups was found only
for IL-18 (Fig. 3). IL-18 continuously decreased in placebo
group after 3 and 5 months. In Lactobacillus group it maintained
at the same level after finishing bacteria in- take (3 m),
and increased 5 months later (p=0,03). Likely to IL-18, IL-12
slightly increased after 8-month observation in children receiving
probiotics in comparison with placebo group, but without
statistical significance (p=0,06) (Fig. 4). TGF-beta1
maintained at similar levels in both groups during the whole
study (Fig. 5).
Discussion
Different probiotic strains have been shown to be useful in
the treatment of AD in children [7, 13, 18, 19]. As atopic diseases
are characterized by imbalance of Th1/Th2 cytokine
profile, it is believed that probiotics affect the immune system
by enhancing of regulatory and/or proinflammatory cytokies
production and by reducing Th2 cytokine release [2].
In addition, probiotics are able to induce a production of secretory
and circulating Abs which create the first line of defense
against external antigens including allergens.
Our group presented that novel probiotic L. casei and
L. paracasei strains markedly reduced the severity of AD in
infants in randomized placebo controlled study [5]. After
3 months of treatment SCORAD index significantly decreased
only in group receiving probiotics, and in IgE-dependent
allergy was significantly lower in comparison with placebo
group. In vitro analyses of those strains using blood
cell cultures of atopic children have shown that they are potent
inducer of both pro-inflammatory and regulatory cytokines,
and on the other hand they do not trigger pro-allergic
responses [14]. In present in vivo study we have shown that
L. casei and paracasei strains activate both humoral and cytokine
responses in children with CM allergy demonstrating
AD. Bacteria induced production of anti-Lactobacillus Abs
mainly of IgA isotype in gut. Although the level of secretory
IgA Abs increased after finishing of bacteria in-take it
was not statistically significant as compared with placebo
group, and then after 5 months the amounts of Abs decreased
to the level found in placebo group. We supposed that
children response to probiotic bacteria by production of anti-Lactobacillus
Abs, but this humoral activation is limited
at time. Our earlier studies performed on germ-free piglets
associated with non-pathogenic Escheichia coli O86 as well
as with newborn children to whom probiotic strains of E. coli
O83 were given presented that probiotic bacteia induced
production of specific Abs, but this response occurred in
short period after bacteria application [3, 4]. In piglets E. coli
colonization induced production of specific IgA Abs in
gut as early as 4 days after bacteria in-take. The level of tho-
Fig. 3 The level of IL-18 in sera of children receiving Lactobacillus strains
and placebo
IL-18 levels were measured by immunoenzymatic method before bacteria
in-take (0m), just after finishing bacteria receiving (3m) and 5 months later
(8m). *statistical significance between children receiving Lactobacillus strains
and placebo.
Fig. 4 The level of IL-12 in sera of children receiving Lactobacillus strains
and placebo
IL-12 levels were measured by immunoenzymatic method before bacteria
in-take (0m), just after finishing bacteria receiving (3m) and 5 months later
(8m). *statistical significance between children receiving Lactobacillus strains
and placebo.
Fig. 5 The level of TGF-beta1 in sera of children receiving Lactobacillus
strains and placebo
TGF-beta1 levels were measured by immunoenzymatic method before bacteria
in-take (0m), just after finishing bacteria receiving (3m) and 5 months
later (8m). There was no statistical significance between children receiving
Lactobacillus strains and placebo.
se Abs rapidly decreased after 15 days, but increased in sera.
In newborn we observed a significant increase in secretory
IgA Ab level starting from 2 weeks after bacteria in-take,
but after 8 weeks the level decreased although still it was

higher (without statistical significance) than in control groups
of children. This phenomenon is explained by the development
of oral tolerance to intestinal non-pathogenic bacteria
[15].
Lactobacillus casei/paracasei strains induced not only
humoral responses but also activated cytokine production
in children. In in vitro studies they activated blood cells of
children with AD to production of IL-12, IL-18, TNF-alpha
and IFN-gamma in higher amounts than known polyclonal
activator PHA [14]. Simultaneously, Lactobacillus strains inhibited
IL-5 secretion. Now we showed that in vivo they slightly
induced proinflammatory IL-12 and significantly modulated
production of IL-18. We observed that serum IL-18
in placebo group decreased during the whole study and this
decrease correlated with clinical improvement. In placebo
group SCORAD decreased after 3 month and then after
5 months as well [5]. In contrast, in children receiving probiotics
in spite of better clinical improvement than in placebo
group, IL-18 did not decrease. Probiotics activated IL-18
production at the same level after finishing of bacteria in-ta-
References
1. Ceregra A, Kozakova H, Czarnowska
E, et al (2005) Effect of gastrointestinal
application of probiotic Lactobacillus
casei/paracasei strains on bacteria translocation
and cytokine production. Folia
Histochem Cytobiol 43 (Supl 1): 28
2. Cukrowska B (2002) The role of the intestinal
microflora in the development
of allergies: the modulation of the immune
system by probiotics. Ann Diag
Paediatr Pathol 2002, 6, 89–96.
3. Cukrowska B, Kozakova H, Rehakova
Z, Sinkora J, Tlaskalova-Hogenova H
(2001) Specific antibody and immunoglobulin
responses after intestinal colonization
of germ-free piglets with non-
-pathogenic Escherichia coli O86. Immunobiology
204: 425–433
4. Cukrowska B, Lodinova-Zadnikova R,
Enders C, Sonnenborg U, Schulze J,
Tlaskalova-Hogenova H (2002) Specific
proliferative and antibody responses
of premature infants to intestinal colonization
with non-pathogenic probiotic
E. coli strain Nissle 1917. Scand J Immunol
55: 204–209
5. Cukrowska B, Piontek E, Najberg E, et
al (2005) The effect of new probiotic
Lactobacillus casei and casei/paracasei
strains on gut ecosystem and clinical
symptoms of children with atopic dermatitis.
Allegy Clin Immunol Intern
(Supl 1): 1645
ke, and even slightly increased its secretion 5 months later.
It was shown that monocytes from patients with AD secreted
reduced amounts of IL-18 [6], and it is known fact that
IL-18 is a potent proinflammatory cytokine able to induce
IFN-gamma, TNF-alpha and IL-1 [10]. This proinflammatory
effect is the result of co-operation with IL-12, which in our
study was slightly higher in children receiving probiotics. On
the other hand IL-18 in the absence of IL-12 induces naive
T-cell into Th2. Thus, IL-18 is a unique cytokine that stimulates
both Th1 and Th2 responses depending on its cytokine
milieu [10]. We supposed that probiotic Lactobacillus casei
and paracasei strains used in our study exert the beneficial
effects in allergic patients by regulation of Th1/Th2 cytokine
profile by IL-18, i.e. cytokine which plays an important
role in the overall immune response.
Acknowledgment
6. Higashi N, Gesser B, Kawana S, Thestrup-Pedersen
K (2001) Expression of
IL-18 mRNA and secretion of IL-18 are
reduced in monocytes from patients
with atopic dermatitis. J Allergy Clin
Immunol 108: 607–614
7. Isolauri E, Matikainen S, Vuopio-Varkila
J, et al (2000) Probiotics in the management
of atopic eczema. Clin Exp
Allergy 30: 1604–1610
8. Kalliomaki M, Salminen S, Arvilommi
H., Koro P, Koskinen P, Isolauri E
(2001) Probiotics in primary prevention
of atopic disease: a randomized placebo-
-controlled trial. Lancet 357: 1076–1079
9. Kalliomaki M, Salminen S, Poussa T,
Arvilommi H, Isolauri R (2003) Probiotics
and prevention of atopic disease:
4-year follow-up of a randomized placebo-controlled
trial. Lancet 361:
1869–1871
10. Nakanishi K, Yoshimoto T, Tsutsui H,
Okamura H (2001) Interleukin-18 is
a unique cytokine that stimulates both
Th1 and Th2 responses depending on
its cytokine milieu. Cytokine Growth
Factors Rev 12: 53–72
11. Pochard P, Gosset P, Grangette C, et al
(2002) Lactic acid bacteria inhibit TH2
cytokine production by mononuclear
cells from allergic patients. J Allergy
Clin Immunol 32: 563–570
12. Pohjavuori E, Viljanen M, Korpela R
(2004) Lactobacillus GG effect in increasing
IFN-gamma production in infants
with cow’s milk allergy. J Allergy
Clin Immunol 114: 131–136
119
This study is supported by the State Committee for Research
(project 2P05E 067 26).
13. Rosenfeld V, Benfeldt E, Nielsen SD,
et al (2003) Effects of probiotic Lactobacillus
strains in children with atopic
dermatitis. J Allergy Clin Immunol
111: 389–395
14. Rosiak I, Witos³aw U, Ceregra A, et al
(2006) The impact of probiotic Lactobacillus
casei and paracasei strains on
cytokine profile in children with atopic
dermatitis. Ann Diag Paediatr Pathol
10: 37–42
15. Tlaskalova-Hogenova H, Stepankova
R, Hudcovic T, et al (2004) Commensal
bacteria (normal mikroflora), mucosal
immunity and chronic inflammatory
and autoimmune diseases. Immunol
Lett 93: 97–10
16. (2005) Probiotic effects on faecal inflammatory
markers and on faecal IgA
in food allergic atopic eczema/dermatitis
syndrome infants. Pediatr Allergy
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17. of inflammation as a possible mechanism
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18. Viljanen M, Savilahti E, Haahtela T, et
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Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 121–124
© Copyright by Polish Paediatric Pathology Society Annals of
Cervical approach to anterior mediastinal tumors in children
Miros³aw M. Krysta 1 , Wojciech J. Górecki 1 , Adam M. Bysiek 1 ,
Witold H. Mie¿ynski 2 , Krzysztof A. Solecki 1 , Bartosz J. Bogusz 1
1 Department of Pediatric Surgery
2 Department of Pathology
The Children's Hospital of the Jagiellonian University
Cracow, Poland
Introduction
Abstract
Mediastinal tumors show many kinds of histology including
malignancy, thus surgery is indicated in almost all cases with
the exception of malignant lymphomas [6]. Recent advances
in thoracoscopic surgery have dramatically altered the approach
to intrathoracic lesions in pediatric patients [8]. Most tumors
found in anterior superior mediastinum are derived
from the thymus and are usually benign [5]. The anterior superior
mediastinum is accessible through a cervical approach
that presents a less invasive alternative to thoracoscopy in
obtaining a tissue diagnosis and resecting thymic tissue [1].
The aim of our study was to assess the safety and effectiveness
of the cervical approach in 11 children with tumors of
the anterior superior mediastinum.
Patients and methods
Between 1996 and 2005, eleven children (8 boys and 3 girls)
aged 1 to 18 years (mean 12) were referred to our service for
Address for correspondence
Recent advance in thoracoscopy reduce the need for thoracotomy for pediatric patients with mediastinal
tumors. The aim of our study is to evaluate the safety and effectiveness of a minimally invasive cervical
approach to anterior mediastinal tumors. A case series of 11 pediatric patients with anterior mediastinal
tumors treated through a minimally invasive cervical approach. Total thymectomy was performed in
8 children and near total thymectomy in 3. Three children required partial sternotomy for exposure. All
wounds healed primary without postoperative complications. All final histological reports matched
intraoperative findings. Persistent thymus was found in 5, thymic hyperplasia in 3, thymic degeneration in
2 and atrophy of thymus in 1 patient. We conclude that the transcervical approach to the anterior
mediastinum is minimal invasive, safe and effective. Thymus may persist beyond the preschool age and
constitutes for clinical dilemma. Thymic hyperplasia may confuse assessment in oncological patients.
Key words: mediastinal tumor, persistent thymus, transcervical thymectomy
Wojciech Górecki, MD voice: +48126580232
Department of Pediatric Surgery fax: +48126581325
The Children’s Hospital of the Jagiellonian University mobile: +48602756661
265 Wielicka St. e-mail: migoreck@cyf-kr.edu.pl
30-663 Kraków, Poland
Diagnostic
Paediatric
Pathology
histological verification of a tumor found in the anterior superior
mediastinum. Seven children had primary tumors, and
four presented with a possible residual mass after treatment
of mediastinal lymphoma. The diagnosis was established by
CT scan in 9 children and MRI in 2.
Surgery was performed under general anesthesia with
endotracheal intubation in the supine position. A transverse
skin incision was made above the sternum. The platysma was
incised horizontally and dissection was advanced vertically
to pretracheal space. The lower border of the wound with
manubrium was raised and dissection proceeded behind the
upper border of the sternum into the retro-sternal space. The
tissue in the superior anterior mediastinum was dissected
with intention of removal. If necessary, a vertical upper sternotomy
extending to the 3rd intercostals space was performed
to optimize exposure. Histological assessment of all tissue
specimens was undertaken intra-operatively. If the mass
was diagnosed as normal thymus tissue and a total thymectomy
would have required extensive exposure, the thymic
remnant was left in place. Residual tumor after treatment for

122
mediastinal lymphoma was removed if possible but biopsied
and left in place if removal would have required extensive
dissection. After hemostasis was achieved, all wounds were
closed without a drain.
Results
Three children required partial sternotomy to optimize exposure.
Thymic tissue was found in all children. Total thymectomy
was performed in 8 and near total in 3 children. A residual
mass was found in 3 of 4 children with lymphoma, and
thymic hyperplasia in one. In 2 of them the mass was excised,
and in the third child it was left in place after a negative
intraoperative biopsy. All final histological reports matched
the macro and microscopic intraoperative findings. The
final diagnoses were summarized with consecutive references
to examples of radiological images (Table 1). The patient
with thymic hyperplasia and unilocular thymic cyst is a very
unique entity (Fig. 1). Scharifker claimed in 2006 that
such case has not been previously published [9]. All wounds
healed primary without postoperative complications. Two
children had repeated thoracic CT scans at 1 and 3 months
postoperatively for a mediastinal hematoma of the retrosternal
space that resolved spontaneously.
Discussion
Video-assisted thoracoscopic surgery, median sternotomy
and the transcervical approach can achieve the same degree
of radical thymectomy [11]. The latter technique is technically
easier, quicker and less invasive.
Half of the children in our series had a normal persistent
thymus, mostly found incidentally. Recognition of po-
Table 1
Diagnosis in 11 patients with anterior mediastinal mass
Macroscopic Final histological Clinical Number Example of
intaoperative findings report diagnosis of patients clinical diagnosis
Normal looking thymic
tissue
Normal thymic tissue Persistent thymus 5 (2*#) Fig. 1
Enlarged fragile thymic
tissue
Normal thymic tissue Thymic hyperplasia 2 (1*) Fig. 2
Enlarged fibrotic Focal lymphatic Thymic degeneration 2 Fig. 3
thymus or cystic degeneration
Enlarged thymus Normal thymic tissue Thymic hyperplasia 1 Fig. 4
with unilocular cyst with unilocular cyst
Atrophic thymus Fibrosous tissue Atrophy of thymus 1*# Fig. 5
* – patients suspected to have residual tumor after mediastinal lymphoma
# – residual tumor found – no active process detected
Fig. 1 MRI of 15 year old boy with persistent thymus
Fig. 2 CT scan of 1 year old boy with thymic hyperplasia

Fig. 3 CT imaging of thymic degeneration in 5 year old boy
Fig. 4 CT image of thymic hyperplasia with unilocular cyst in 5 year old boy
ssible persistence and normal variation in the CT appearance
of the thymus in young patients may prevent the false-positive
diagnosis of a neoplasm [4].
One patient in the series had thymic hyperplasia after
chemotherapy for Hodgkin disease, a common finding that
occurs in more than one third of patients with thymic involvement
in Hodgkin’s lymphoma despite a full clinical remission
after treatment [13]. Moreover, thymic hyperplasia
is well known as a potential differential diagnosis of media-
References
1. Crucitti F, Zucchetti F, Doglietto GB
(1977) The cervical approach in surgery
of the thymus for myasthenia. Comparison
with the trans-sternal route.
Considerations on 75 operated patients.
Minerva Chir 32: 543–547
stinal space-occupying lesions and also as a long-term complication
in patients cured of Hodgkin’s disease [10]. This
may raise doubt concerning possible tumor recurrence [2].
An evolving alternative to transcervical biopsy and
resection of a thymic mass may be percutaneous CT-guided
needle biopsy, which allows access to the lesion in virtually
all mediastinal locations [3]. This may indeed become the
method of choice for the study of mediastinal tumors in the
future [7]. However, in one of six patients an image-guided
needle biopsy does need yield adequate tissue for diagnosis,
and therefore the transcervical approach will be indicated in
these cases [12].
Conclusions
2. Cumin I, Mechinaud F, Harousseau JL
(1996) Thymus hyperplasia following
chemotherapy. Presse Med 25: 291–292
3. Gupta S, Seaberg K, Wallace MJ, et al
(2005) Imaging-guided percutaneous
biopsy of mediastinal lesions: different
approaches and anatomic considerations.
Radiographics 25: 763–786
123
Fig. 5 Chest X-ray of 18 year old girl with residual tumor after HL. Atrophic
thymus and residual tumor were subsequently removed
The transcervical approach to the anterior mediastinum is
minimal invasive, safe and effective. Thymus may persist
beyond the preschool age and constitutes for clinical dilemma.
Thymic hyperplasia may confuse assessment in oncological
patients.
4. Heiberg E, Wolverson MK, Sundaram
M, Nouri S (1982) Normal thymus: CT
characteristics in subjects under age 20.
AJR Am J Roentgenol 138: 491–494
5. Maruszynski M, Plachta H, Kott M, Obara
A (1994) Surgical treatment of mediastinal
tumors from personal material.
Pneumonol Alergol Pol 62: 491–495

124
6. Masaoka A (1985) Treatment of mediastinal
tumor. To Kagaku Ryoho 12:
1383–1391
7. Romero-Guadarrama MB, Duran-Padilla
MA, Cruz-Ortiz H, et al (2004)
Diagnosis of thymolipoma with fine needle
aspiration biopsy. Report of a case
initially misdiagnosed as liposarcoma.
Acta Cytol 48: 441–446
8. Rothenberg SS (1998) Thoracoscopy in
infants and children. Semin Pediatr
Surg 7: 194–201
9. Scharifker D (2006) True thymic hyperplasia
associated with a unilocular thymic
cyst: an unusual combination not
previously reported. Ann Diagn Pathol
10: 32–35
10. Scheinpflug K, Schmitt J, Jentsch-Ullrich
K, Roessner A, Franke A (2003)
Thymic hyperplasia following successful
treatment for nodular-sclerosing
Hodgkin’s disease. Leuk Lymphoma
44: 1615–1617
11. Seguier-Lipszyc E, Bonnard A, Evrard
P, Garel C, De Ribier A, Aigrain Y, de
Lagausie P (2005) Left thoracoscopic
thymectomy in children. Surg Endosc
19: 140–142
12. Shabb NS, Fahl M, Shabb B, Haswani
P, Zaatari G (1998) Fine-needle aspiration
of the mediastinum: a clinical, radiologic,
cytologic, and histologic study
of 42 cases. Diagn Cytopathol 19:
428–436
13. Wernecke K, Vassallo P, Rutsch F, Peters
PE, Potter R (1991) Thymic involvement
in Hodgkin disease: CT and sonographic
findings. Radiology 181:
375–383

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 125–128
© Copyright by Polish Paediatric Pathology Society Annals of
Rare case of calcyfying fibrous pseudotumour
of the lung in 6 years old girl
Przemys³aw Przewratil 1 , Anna Sitkiewicz 1 , Józef Kobos 2 , Ewa Andrzejewska 1
1 Department of Pediatric Surgery and Oncology
2 Department of Pathology of Age of Development
Medical University of Lodz
Introduction
Abstract
Inflammatory tumors (inflammatory myofibroblastic tumor
– IMT) are rare benign tumors mainly in children and youth.
About 20% of cases are accompanied by fever, body weight
loss and progressing anaemia. They are present mainly in
a lung or abdomen, however, they might also be in other organs
[2, 3, 6]. Their clinical course may even suggest a malignant
neoplasm due to its local invasiveness and tendency
to recurrence (10–25%). They may also metastasize (about
5%) or become malignant. Sizes of this tumor are varied
and depend on its localization; usually they vary between
5–10 cm. Histologically, they consist of miofibroblasts, lymphocytes,
plasmatic and mesynchymal cells [1, 2, 3]. Not in-
Address for correspondence
Six-year old girl was addmited to the surgery department with diagnosis of thoracic tumor. CT scans
demonstrated solid tumor of the right thorax. Increased inflammatory index and distinct anaemia were
detected in laboratory tests. On the basis of CT imaging and neoplastic markers the proper diagnosis of the
tumor characteristics was not possible. The child was qualified for surgery with suspition of mature
teratoma. The giant solid tumor of the right lung, tightly attached to diaphragm was found during
thoracotomy. Tumor was excised completly within right lung. Histopathological examination revealed
inflammatory myofibroblastic tumor (IMT) of the lung with characteristic proliferation of myofibroblasts
with definite infiltrations of chronic inflammatory cells. In central part of the tumor excessive bone
metaplasia fields and calcifications, focally with fields of high cellularity were identified.
Immunohistochemical tests were positive for SMA, CD20 and negative for desmine, MC, Alk-1 and p53.
These results with some doubts lead to diagnosis of very rare type of calcifying fibrous pseudotumor (CFP).
The girl had progression of microscopic residual mass in the chest 6 months after surgery and needed
steroidotherapy with good result. The review of literature about IMT and CFP in children is also presented
in this paper.
Key words: calcifying fibrous pseudotumor, inflammatory tumor, lung tumor
frequently, they pose diagnostic difficulties. The basis of an
effective treatment is a complete excision of the lesion. In selected
cases, an aggressive supplementary therapy is also taken
into consideration [7].
Case report
Przemys³aw Przewratil, MD, PhD tel./fax (42) 61-77-705, 504 271 279
Department of Pediatric Surgery and Oncology e-mail: goral3@interia.pl
Medical University of Lodz
ul. Sporna 36/50,
91-738 £ódŸ
Diagnostic
Paediatric
Pathology
For many months, a 6-year old girl suffered from recurring
respiratory system infections. Since the infection appeared
again, a girl was admitted to the pediatric ward. A lungs
X-ray showed an immense chest tumor. Moreover, a girl had
a high fever and she had progressive anaemia. CT confirmed
the presence of a solid tumor which took up two thirds of the
right side of chest (Fig. 1). Additional tests showed high acu-

126
Fig. 1 CT scan before surgery shows involvement of the whole right chest
by giant neoplastic tumor
te phase indicators and progressive anaemia requiring blood
transfusion. After the broad spectral antibiotic therapy was
applied, CRP was lowered. On the basis of image examination
(USG, CT) and neoplastic markers (levels of catecholamines,
ferritin, LDH, AFP, β-HCG) it was impossible to define
the type of tumor. A child was qualified for surgery with
the preliminary diagnosis of mature teratoma (in X-ray examination
– calcification within the tumor). During the right-
-side thoracotomy a solid hard tumor was found, tightly joined
with diaphragm and embracing a lower and middle lung
lobe. After the partial dissection and cutting the tumor, it was
found that a vascular lung stalk was affected and that it was
closely joined with pericardium. A tumor was completely removed
together with the right lung (Fig. 2) and drainage to
pleural cavity was inserted.
Fig. 2 Inflammatory tumor of the righ lung after removal. Intact part of
pulmonary tissue in the upper lobe is visible
Macroscopically, a white-grayish elastic tumor, infiltrating
almost the whole lung, with no necrosis or hemorrhage
foci was described. A tumor was partially surrounded by
a thin capsule and did not exceed the resected lung tissues.
In the medial part, a tumor had hardness and consistency of
a bone. Microscopically, findings obtained revealed that a tumor
mainly consisted of bundles of spindle-shaped cells with
myofibroblastic morphology (Fig. 3). In some preparations,
a rich infiltration from lymphoid, plasmatic cells and granulocytes.
Focally, intensified fibrosis, hyalinization and calcifications
were revealed. In some preparations, vascular and
pericyte proliferation as well as myofibroblast proliferation
in follicles were revealed. Focally, big non-typical cells with
distinct nucleolus also were observed. In some preparations,
tumor texture revealed a high “cellularity” with osteoplasia
fields. In peripheral tissues, in macroscopically unchanged
lung, there was no tumor infiltration. The examination also
indicated that the tumor excision was complete. Moreover,
a series of immunohistochemical tests revealed a strong
expression of SMA in proliferation cells and CD20 in most
of the mononuclear cells. No expression of desmine, MC,
Alk-1, p53 in the tumor cells was revealed.
Fig. 3 Tumor specimen mainly consisted of bundles of spindle-shaped cells
with myofibroblastic morphology
In summary, a histopathologic examination revealed
a rare type of inflammatory tumor (inflammatory myofibroblastic
tumor) with excessive bone metaplasia fields and calcifications,
focally with fields of high cellularity (Fig. 4).
In the postoperative course, no significant postoperative
complications were reported. As a result of further treatment,
including 6 days in an intensive care ward (breathing
support, analgosedation, broad spectral antibiotic therapy,
blood transfusion) normalization of acute phase indicators
and blood morphology was achieved. On the 18th day after
operation, a patient with full respiratory function was discharged
and further outpatient treatment was recommended.
PET (positon emission tomography) examination
carried out 6 months after operation revealed residual lesion

Fig. 4 Specimen from central part of tumor with excessive bone metaplasia
fields and calcifications, focally with fields of high cellularity
in the right part of the chest. Therefore, immunosuppressive
treatment with steroids was launched (Encorton for one
month). PET monitoring examination carried out 10 months
later revealed regression of the above mentioned lesion. Up
till now, a girl develops correctly, does not show any respiratory
system disorders or immune deficiencies (2 years from
operation).
Discussion
A first description of an inflammatory tumor comes from
1937 and since then, about 300 cases of his tumor in children,
in different localizations, were reported. Most often, it is located
in lungs and it affects one organ. In the English-language
academic literature, one can come across diversified nomenclature,
among others plasma cell granuloma, inflammatory
pseudotumor, and most often used – IMT [2, 7].
Reasons for the inflammatory tumor occurrence are
still unclear. According to one of the theories, it is a typical
locally aggressive neoplastic tumor. Recent research on the
limited groups of patients revealed the presence of the clonal
cytogenetic disorders of ALK (anaplastic lymphoma kinase)
gene on 2p chromosome and the ALK protein expression,
similarly to anaplastic gigantocellular lymphoma. The
clinical course also suggests the neoplastic character of the
tumor [2, 11]. According to other theory, the formation of
these pathologic masses results from immunological reactions
to infections or other stimulatory factor such as operations,
injuries or exposure to radiation [1, 6, 7]. Mycobacterial
origin of the inflammatory tumors was also reported, i.e.
in patients with HIV infection. Bacterial structures in the tumor
cells, visible in an electron microscope examination may
also suggest infectional origin [2, 4, 10].
An inflammatory tumor poses diagnostic difficulties
since it does not provoke the increase in neoplastic markers
in serum. Radiological image of this tumor is also difficult
to diversify. CT may properly locate the lesion but it does not
explain its character [1, 6]. In the case of our patient, the neo-
127
plastic process was considerably advanced and it affected the
two thirds of the chest; the tumor had numerous calcifications,
which, in the absence of positive neoplastic markers,
could suggest mature teratoma. A basis for diagnosis is therefore
a histopathologic examination of the whole removed
tumor.
A characteristic feature of inflammatory tumors is the
proliferation of fibroblasts and myofibroblasts with definite
infiltrations of chronic inflammatory cells, in particular, plasmatic
cells. This differentiates them from fascitis or fibromatosis.
Exceptionally, necrosis foci or calcifications may
occur, as well as cytomorphologic malignancy features [2, 3,
5, 11].
There are three histological types which may occur
within one tumor with different predominance: 1) myxoidal/vascular
type, resembling granulation inflammatory tissues,
2) spindle-shaped type with fields of different cellular
density, 3) hypocellular pattern densely collagenized and reminiscent
of fibrous scar [3]. In the presented case, a histopathological
image mostly corresponded to the given characteristics,
however focally there were changes of different
kind.
In immunohistochemical examinations, inflammatory
tumors reveal positive reaction to SMA (smooth muscle actin
– 92%) and MSA (muscle specific actin – 89%) and, to
a lesser degree, to desmin (69%) and keratin (36%). It confirms
the myofibroblastic phenotype of the spindle-shaped
cells. Recent findings also reveal the ALK protein overexpression
in the tumor cells [3, 10, 11]. In the discussed patient
a strong SMA expression in proliferative cells and
CD20 in most mononuclear cells was found. However, no
expression of desmin, MC, Alk-1, p53 in the tumor cells was
found. A high heterogenicity of the inflammatory tumors goes
together with their different degree of aggressiveness. Up
till now, no definite histological criteria to forecast the clinical
course of the tumor have been determined. Cytogenetic
research determining the tumor’s DNA might be helpful. It
seems that the combination of cellular atypia, occurrence of
ganglion-resembling cells, p53 expression and DNA aneuploidia
may indicate the potentially aggressive tumor type
with the increased tendency to recurrence and maliganat
transformation [7, 10, 11].
When suspecting the malignant proliferation in differential
diagnostics one needs to consider the soft tissues tumors,
including inflammatory malignant fibrous histiocytoma,
sarcomatoid carcinoma, angiosarcoma, leiomyosarcoma,
fibrosarcoma. RMS. It might also be a direction of a potential
transformation into a malignant type of an inflammatory
tumor [2, 7].
A discussed case of a lung tumor in a 6-year old girl
deserves a particular attention since in its central part, specific
morphological changes were found. These changes let us
qualify this tumor as a special type of inflammatory tumor,
calcifying fibrous pseudotumor (CFP). It is an extremely rare
tumor, histologically classified as a separate group. It is
characterized by a considerable hyalinization and dystrophic
calcification. This type of changes may locally recur, but the

128
distant metastases were not reported [3, 8, 12]. There are few
reports of this type of tumor. They affect neck, digestive
tract, mediastinum and pleural cavity [8]. Reports of this type
of tumor located in a lung appeared for the first time in
the medical academic literature in the years 2003 and 2004
[9, 12]. We might therefore presume that the presented case
is one of the few documented cases in the world.
A type of calcifying tumor is histologically characterised
by hypocellular hyalinized collagenous tissue with
psammomatous and/or dystrophic calcifications and patchy
lymphoplasmacytic infiltrates, however, the calcifications
might have different advancement degree. In the presented
case, focally, intense fibrosis and hyalinization as well as calcifications
were visible. In immunohistochemical examinations
CFP indicates a positive reaction with vimetin, CD34
and XIIIa factor, but a negative reaction with epithelial membrane
antigen, keratin, SMA, desmin, S-100 protein and
ALK [8, 9, 12, 13]. In the presented case, a reaction with
SMA was intensely positive and a reaction with ALK and desmin
negative. It is therefore difficult to interpret that unam-
References
1. Cerfolio RJ, Allen MS, Nascimento
AG, Deschamps C, Trastek VF, Miller
DL, Pairolero PC (1999) Inflammatory
pseudotumors of the lung. Ann Thorac
Surg 67: 933–936
2. Dehner LP (2000) The enigmatic inflammatory
pseudotumours: the current
state of our understanding, or misunderstanding.
J Pathol 192: 277–279
3. Fletcher CDM (2001) Soft tissue tumors
In: Fletcher CDM Diagnostic Histopathology
of Tumors, Churchil Livingstone,
London, pp1500–1501
4. Gale N, Zidar N, Podboj J, Volavsek M,
Luzar B (2003) Inflammatory myofibroblastic
tumour of paranasal sinuses
with fatal outcome: reactive lesion or
tumour? J Clin Pathol 56: 715–717
5. Hill KA, Gonzales-Crussi F, Chou PM
(2001) Calcifying fibrous pseudotumor
versus inflammatory myofibroblastic
tumor: a histological and immunohistochemical
comparison. Mod Pathol 14:
784–790
6. Hoer J, Steinau G, Fuzesi L, Gunawan
B, Schumpelick V (1999) Inflammatory
pseudotumor of the diaphragm. Pediatr
Surg Int 15: 387–390
7. Karnak I, Senocak ME, Ciftci AO, Caglar
M (2001) Inflammatory myofibroblastic
tumor in children: diagnosis and
treatment. J Ped Surg 36: 908–912
8. Nascimento AF, Ruiz R, Hornick JL,
Fletcher CD (2002) Calcifying fibrous
“pseudotumor”: clinicopathologic study
of 15 cases and analysis of its relationship
to inflammatory myofibroblastic
tumor. Int J Surg Pathol 10: 189–196
9. Peachel M, Mayo J, Kalloger S, Flint J,
English J (2003) Calcifying fibrous
pseudotumour of the lung Thorax 58:
1018–1019
10. Sarker A, An C, Davis M, Praprotnik D,
McCarthy LJ, Orazi A. (2003) Inflammatory
pseudotumor of the spleen in
a 6-year-old child: a clinicopathologic
study. Arch Pathol Lab Med. 127:
e127–130
biguously. Most probably, expression of the examined proteins
changes in the individual parts of the tumor.
There is a theory that CFP is a final stage of the inflammatory
tumor advancement [5, 12, 13]. In our patient,
a lung tumor was enormous, affected two thirds of the chest,
which indicates that the disease course lasted for many months,
maybe even years, which could support this theory. Some
hypotheses claim that the discussed proliferation is
a completely different tumor from IMT. The grounds for
such speculations is the lack of ALK and SMA protein in
CFP tissues, as well as the fact that IMP rarely contains calcifications
[5, 13].
Upon the analysis of the histological material and immunohistochemical
results of the removed tumor, there are
three speculations: 1. this tumor is a CFP with non-typical
positive SMA expression, 2. this tumor is a mixed form, including
both IMT and CFP components, 3. it is an inflammatory
tumor, which due to a long development period, was
subject of calcification, characteristic for CFP, in the central
part.
11. Sastre-Garau X, Couturier J, Derre J,
Aurias A, Klijanienko J, Lagace R
(2003) Inflammatory myofibroblastic
tumour (inflammatory pseudotumour)
of the breast. Clinicopathological and
genetic analysis of a case with evidence
for clonality. J Pathol 200: 269–271
12. Soyer T, Ciftci AO, Gucer S, Orhan D,
Senocak ME (2004) Calcifying fibrous
pseudotumor of lung: a previously
unreported entity. J Ped Surg 39:
1729–1730
13. Van Dorpe J, Ectors N, Geboes K,
D’Hoore A, Sciot R (1999) Is calcifying
fibrous pseudotumor a late sclerosing
stage of inflammatory myofibroblastic
tumor? Am J Surg Pathol 23: 329–335

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 129–131
© Copyright by Polish Paediatric Pathology Society Annals of
Malignant fibrous histiocytoma at 6 years old boy
– a case report
Micha³ Rólski 1 , Jerzy Harasymczuk 1 , Przemys³aw Mañkowski 1 ,
Andrzej Jankowski 1 , Pawe³ Kroll 1 , Ma³gorzata Warzywoda 2 , Ewa Trejster 3
1 Department of Pediatric Surgery, Traumatology & Urology
2 Department of Pediatric Radiology
3 Department of Pathomorphology
Poznan University of Medical Sciences
Poland
Introduction
Malignant fibrous histiocytoma (MFH) of the gastrointestinal
tract – primary and metastatic is uncommon, even though
it is most frequently diagnosed malignant soft tissue tumor
in adults. In the world literature only 22 cases of MFH
of the gastrointestinal tract were described, but only in adult
patients [5, 10, 11]. That is a reason, why we present MFH
of stomach in 6-year-old boy.
Case report
Abstract
Six years old boy was admitted to Department of Pediatric
Surgery, Traumatology & Urology, Poznan University of
Medical Sciences from regional hospital because of considerable
anemia caused by bleeding from gastrointestinal tract
and bronchopneumonia. The child was pale; we observed
loss of weight and melena.
In initial ultrasonography (USG), a solid hypogenic
homogenous mass was visible around the subphrenic part of
the esophagus and close to the cardia and posterior wall of
the stomach. There were no calcifications. Subsequent USG
examinations with color and Power Doppler showed multifocal,
solid, vascularized mass surrounded with slightly en-
Address for correspondence
Authors present a case of very rare primary neoplasm of stomach – malignant fibrous histiocytoma at
6 years old boy. Surgical procedures were performed with a good therapeutic result.
Key words: children, malignant fibrous histiocytoma, stomach
Jerzy Harasymczuk, MD, PhD Phone: 061 8475228
Department of Pediatric Surgery, Traumatology & Urology E-mail: harasymczuk@o2.pl
Poznan University of Medical Sciences
Szpitalna 27/33
60-572 Poznañ
Diagnostic
Paediatric
Pathology
larged lymph nodes (1,5 cm). Sizes of the tumors were
4,0 × 2,5 × 3,5 cm; 4,0 × 3,3 × 3,5 cm and 1,3 × 1,8 cm. All
other abdominal organs were normal in USG. There was no
free fluid in peritoneum cavity (Fig. 1).
A gastrointestinal passage (of the esophagus and the
stomach) was performed at the same time. It showed winding
and slight narrowing esophagus in its subphrenic part,
without any disturbances of a contrast medium passage.
Fig. 1 Ultrasound scan of abdomen shows gastric tumor

130
There was irregularity of the stomach margin in the cardia
and fundus region similar to filling defect. It suggested an
extra- or intramural mass of the stomach and/or esophagus
(Fig. 2).
An abdominal CT scan showed an asymmetrical thickening
of the esophageal walls on phrenic level. A lumen in
this part of the esophagus was slit-like. The infiltration involved
the cardia and the lesser curvature of the stomach on the
serous membrane side. The size of this irregular unhomogenous
solid mass was 4.8x3.8 cm. Some of the lymph nodes
were visible around. The liver, spleen, pancreas, kidneys and
adrenal glands were normal. The origin of the mass seemed
to be the wall of the stomach (Fig. 3).
Due to growth of the tumor and appearance of metastases
on back wall of stomach a decision about surgical treatment
was made. Anterior stomach wall was opened and large
tumor descending from antrum and minor curve was
Fig. 2 A gastrointestinal passage preoperative study
Fig. 3 Abdominal CT of 6-year-old boy
found. A neoplasm filling fundus and gastric trunk was observed.
The biopsy was taken for histopathology examination
and a gastrostomy was performed. Soon after the operation
gastrostomy was blocked by tumor tissues. According to histopathology
radical surgical treatment was performed. Resection
of intraabdominal part of esophagus, cardia, and stomach
trunk together with nearby nodes and spleen was performed.
Remaining part of a stomach wall was attached with
single stitches to the distal part of esophagus. Next pyloroplasty
according to Mikulicz was performed. No complications
were observed in the postoperative course. Oral feeding
was started on 6. postoperative day. Until today – the boy is
still doing well.
Discussion
In the 1978 Weiss and Enzinger [10] described 200 cases
with MFH typically arises in one of the extremities and less
commonly at the retroperitoneal space of abdomen. This largest
review showed that this tumor is most common between
50-70 ages and 70% of patients are male.
In our opinion and experience from diagnostic point
of view MFH localized in the stomach and the esophagus is
very difficult to recognize with only one imaging method [7].
We have used three of them: contrast enema, USG and CT.
Each of them has provided us with important information for
clinical point of view. The first imaging should be ultrasonography,
as usual in case of abdominal problems signs. We
suggest that the next method may be contrast enema because
of its value in functional assessment of the digestive tract.
CT scan was very important for preoperative planning for
surgical surgical approach.
Adjuvant chemotherapy remains controversial for
soft tissue sarcomas, with some randomized clinical studies
showing a clear advantage in overall or disease free survival
rate [2, 3, 6, 9], whereas other show no benefit of adjuvant
chemotherapy [1, 4, 8].
Conclusions
Clinical follow-up examination conducted every 3 months
showed good general condition, increase of body weight and
normalization of laboratory data. Ultrasonography and contrast
X-ray examination showed no symptoms of metastases
or local expansion of the tumor. Because of various opinions
about chemotherapy in such cases, we decided to restrict the
treatment to surgery with continuous monitoring of clinical
state including regular USG and laboratory examinations.
Patient after two years of follow-up remains in a good general
condition.

References
1. Alvegard TA (1986) Adjuvant chemotherapy
with Adriamycin in high-grade
malignant soft-tissue sarcoma: a Scandinavian
randomized study. Proc Am
Soc Clin Oncol 5: 485
2. Benjamin RS, Terianian TO, Fenogilo
CJ, et al (1987) The importance of combination
chemotherapy for adjuvant treatment
of high-risk patients with soft-
-tissue sarcomas of the extremities. In:
Salomon SE (ed) Adjuvant Therapy of
Cancer. Grune and Stratton, Orlando,
pp 734–744
3. Chang AE, Knisella T, Glatstein E, et al
(1988) Adjuvant chemotherapy for
patient with high-grade soft-tissue sarcomas
of extremity. J Clin Oncol 6:
1491–1500
4. Edmonson JH (1985) Systemic chemotherapy
following complete excision of
nonosseous sarcomas. Mayo Clinic
Experience 3: 89–107
5. Enzinger FM, Weiss SW (1988) Soft
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Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 133–136
© Copyright by Polish Paediatric Pathology Society Annals of
Umbilical remnant abnormalities: a review of 5 cases
Ma³gorzata Pacholska 1 , Ma³gorzata Chrupek 1 , Irena Daniluk-Matraœ 1 ,
Przemys³aw Ga³¹zka 1 , Magdalena Chrzanowska 1 , Roman KaŸmirczuk 2 ,
Piotr Brzeziñski 2 , Sylwia Drewa 3 , Zdzis³aw Skok 4 , Andrzej Igor Prokurat 1
1 Department of Pediatric Surgery
2 Department of Anaesthesiology and Intensive Care
3 Department of Radiology
4 Department of Pathology
Nicolaus Copernicus University
Collegium Medicum in Bydgoszcz, Poland
Introduction
Abstract
Umbilical cord anomalies in the neonate include abdominal
wall defects, omphalomesenteric duct remnants, and urachal
remnants [7]. The urachus is a vestigial fibromuscular tube
from the apex of the bladder to the umbilicus, whereas patent
omphalomesenteric duct is a fistula between the ileum
and the umbilicus. Omphalomesenteric duct and urachal
remnants may persist due to patency of the entire structures
or some portion of it. Four major variants are described in
most series: sinus, cyst, diverticulum, and patent omphalomesenteric
duct or patent urachus respectively [8]. Correct
differential diagnosis between these anomalies is usually made
after careful clinical examination as well as assessment of
discharged umbilical fluid in laboratory tests. Recently ultrasound
examination has emerged as the study of choice in delineating
these anomalies [12].
Only a few cases of persistent patency of both yolk
sac remnants have been reported in the literature [6]. We report
five cases of various umbilical remnants abnormalities
Address for correspondence
Umbilical cord anomalies in the neonate include abdominal wall defects, omphalomesenteric duct remnants,
and urachal remnants. Correct differential diagnosis between these anomalies is sometimes difficult and
usually made due to careful clinical examination as well as assessment of discharged umbilical fluid in
laboratory tests. Only a few cases of persistent patency of both yolk sac remnants have been reported in
the literature. We report five cases of various umbilical remnants abnormalities in newborns, in one
combined with patency of omphalomesenteric duct and the presence of the urachal sinus.
Key words: patent omphalomesenteric duct, umbilical cord anomalies, urachal remnants
Ma³gorzata Pacholska Phone: 052 585 40 15
Department of Pediatric Surgery Fax: 052 585 40 95
Nicolaus Copernicus University E-mail: kikchirdz@cm.umk.pl
Collegium Medicum in Bydgoszcz
Sklodowskiej-Curie 9, 85-094 Bydgoszcz, Poland
Diagnostic
Paediatric
Pathology
in newborns treated in the Department of Pediatric Surgery
in Bydgoszcz, between October 2004 and January 2005, in
one case combined with patency of omphalomesenteric duct
and the presence of the urachal sinus.
Case 1
A 3-day-old boy, product of uneventful vaginal delivery in
39 HBD, presented with umbilical cord discharge. Careful
physical examination showed fistula in the umbilicus with
fecal discharge exuded through this fistula. Gram stain and
culture of the fluid were negative for infection. Periumbilical
area also seemed to be free of infection. Baby was referred
for the operation. At surgery, a supraumbilical transverse
incision was made exposing patent omphalomesenteric
duct which was resected with the small segment of bowel
and the ileum was anastomosed (Fig. 1). Pathologic analysis
of the umbilical cord confirmed the presence of ileal-jejunal
type of mucosa without heterothopic tissue. The child’s postoperative
recovery was uneventful.

134
Fig. 1 Patent omphalomesenteric duct in 3 day-old neonate /case 1/
Case 2
A baby boy was born at 34 weeks’ gestation, the delivery
was uneventful. At the age of 24 days he was referred to our
clinic from an outside institution. Since 3 weeks after patient’s
birth, his parents noticed mild local swelling of the
umbilicus and periodically small amount of a yellow fluid
drainage from the umbilicus, similar to the patient’s urine.
Physical examination showed small, thick fistula in umbilicus.
Diagnosis based on ultrasound and fistulography revealed
persistent urachus. Patient was operated on when he was
30 days-old. The patent urachus was resected and the bladder
was sutured (Fig. 2). Histopathology revealed typical
bladder mucosa. No other anomalies were noted. Postoperative
voiding and ultrasonography of bladder and kidneys were
normal.
Fig. 2 Patent urachus in 3 week-old neonate /case 2/
Case 3
A full term baby boy with routine prenatal care, but no prenatal
ultrasound examinations, was referred from an outside
institution after an uneventful delivery. The patient was noted
to have an umbilical cord hernia and patent omphalome-
senteric duct. Diagnosis was based on careful clinical examination
(Fig. 3). At laparotomy umbilical cord hernia was repaired
and patent omphalomesenteric duct was identified. Limited
segmental small bowel resection, including pesistent
omphalo-mesenteric duct was completed. The postoperative
specimen histology revealed only intestinal mucosa lining
the fistula, and no gastric mucosa was identified. The child’s
postoperative recovery was uneventful.
Fig. 3 Umbilical cord hernia and patent omphalomesenteric duct seen within
the hernia in full term neonate /case 3/
Case 4
A baby boy was born at 35 weeks’ gestation. At the age of
3 days he was referred to our clinic from an outside institution
because of umbilical cord discharge. Careful clinical
examination revealed two umbilical fistulas and moderate
skin irritation around umbilicus. Umbilical cord had been
moist from continued fluid and gases drainage from the upper
umbilical fistula. No other anomalies were noted. Ultrasound
examination suggested also urachal anomaly. For this
reason we performed fistulogram revealing urachal sinus lying
caudal to the upper umbilical fistula (Fig. 4).
At the surgery patent omphalomesenteric duct was
resected with small segment of bowel and the ileum was reanastomosed
(Fig. 5). Urachal sinus was than separated and
resected. Histopathological examination of the operative
specimens revealed normal ileal and urinary mucosa respectively.
Postoperative voiding and ultrasonography of bladder
and kidneys were normal. The child’s postoperative recovery
was uneventful.
Case 5
A 1-day-old male was born at 32 weeks’ gestation after an
uneventful perinatal course. After birth he developed clinical
picture of ileus with bilious vomiting and abdominal distension
(Table 1). He passed also a small amount of pale
meconium. An abdominal x-ray study confirmed an intestinal
obstruction (Fig. 6). Clinical examination revealed two
separated fistulas in umbilicus, with a stool drainage through
one of the fistula. At laparotomy proximal and distal segment

Fig. 4 Fistulogram of urachal sinus lying caudal to the upper umbilical
fistula in 3-day old neonate /case 4/
Fig. 5 Patent omphalomesenteric duct in the same patient /case 4/
Table 1
Summary of clinical features in the whole group of neonates
with omphalomesenteric duct and urachal remnants
Case Leak from Redness Ileal Age at
umbilicus around obstruction surgery
umbilicus /days/
1 + – – 4
2 + – – 30
3 + – – 2
4 + + – 6
5 + – + 2
of small bowel were opened within the umbilicus with limited
patency of proximal loop. No other anomalies were noted.
Following resection of small segments of proximal (moderately
distended) and distal ileum a primary end-to-end ileo-ileal
anastomosis was performed. The postoperative course
was uneventful.
Discussion
135
Fig. 6 Abdominal x-ray of 1 day-old neonate with two separated fistulas in
the umbilicus and the limited patency of proximal loop. Note the radiological
picture of ileus /case 5/
Umbilical discharge in an infant occurs usually due to granulation
tissue, retained umbilical cord elements, infection or
sinuses of either the urachus or the omphalomesenteric (vitelline)
duct [10]. In case of persistent umbilical discharge
not due to infectious granulation tissue, persistent patency of
embryologic remnants must be considered [6]. Establishing
of final diagnosis in such cases on the clinical background
sometimes become difficult.
In our series all hospitalized infants were male and
two of them were born before 36th week of gestation. Although
the combination of disorders of umbilicus are rare three
of five reviewed infants had composite abnormalities of
umbilicus [2]. Two of them had both persistent patent yolk
sac anomalies. All of these children had clinical findings as
a exuded liquid or gases through the fistula in the umbilicus,
hernia of umbilical cord, local swelling or redness around
umbilicus. Those symptoms reviled during the first careful
clinical examination are of high importance in the case of
newborn with suspicion of congenital umbilical anomaly. In
all our cases diagnosis was based on clinical and radiological
findings and was established in all cases before operation
(Table 2). Authors preferred additionally to clinical examination
to perform abdominal ultrasound. This examination
has emerged as a very useful non-invasive diagnostic method
in delineating omphalomesenteric and urachal anomalies [1,
12]. In two of three cases where contrast radiological exami-

136
Table 2
Summary of clinical and radiological confirmations of presence
of omphalomesenteric duct and urachal remnants in
the whole group of neonates
Case Clinical Ultrasono- Fistulo- Intraexamination
graphy graphy operative
diagnosis
1 + – – +
2 + + + +
3 + – – +
4 + + + +
5 + – + +
nations were undertaken, cystography or fistulography were
necessary to visualize urachal patology (Table 2). According
to Lizerbram, ultrasonography may not depict a small patent
urachus and/or omphalomesenteric duct and in these cases he
proposed T2-weighted images due to the high T2 signal intensity
of fluid within both tracts. However, the T1-weighted
gadolinium- enhanced sequence did not add further information
beyond that seen by cystography [6]. In our cases, ultrasonography
performed by skilled radiologist allowed to
visualise urachal remnants quite accurately.
Although few reports discuss the spontaneous regression
of the fistula in case of persistent umbilical remnant, we
recommend surgery as definitive procedure to confirm and
treat these anomalies [4]. Early surgical management is ne-
References
1. Avni EF, Matos C, Diard R, et al (1988)
Midline omphalovesical anomalies in
children: contribution of ultrasound
imaging. Urol Radiol 10: 189–194
2. Boyle G, Rosenberg HK, O’Neill J
(1988) An unusual presentation of an
infected urachal cyst. Clin Pediatr 27:
130–134
3. Kalter CS, Williams MC, Vaughn V, et
al (1994) Sonographic diagnosis of
a large umbilical cord pseudocyst. Am
Inst US Med 13: 487–489
4. Kamii Y, Zaki AM, Honna T, Tsuchida
Y (1992) Spontaneous regression of patent
omphalomesetneric duct: from a fistula
to Meckel’s diverticulum. J Pediatr
Surg 27: 115–116
5. Konvolinka CW (2002) Patent omphalomesenteric
duct. Surgery 131: 689–690
6. Lizerbram EK, Mahour GH, Gilsanz V
(1997) Dual patency of the omphalomesenteric
duct and urachus. Radiol 27:
244–246
7. Lugo B, McNulty J, Emil S (2006)
Bladder prolapse through a patent urachus:
fetal and neonatal features. Journal
of Pediatric Surgery 41: E5–E7
8. McCollum MO, MacNeily AE, Blair
GK (2003) Surgical implications of
urachal remnants: presentation and management.
J Pediatr Surg 38: 798–803
9. Nobuhara KK, Lukish JR, Hartman
GE, Gilbert JC (2004) The Giant Umbilical
Cord: An Unusual Presentation of
a Patent Urachus. J Pediatr Surg 39:
128–129
cessary because congenital umbilical cord anomalies can
present with acute life-threatening complications such as
small bowel prolapse, obstruction, intestinal inflammation/hemorrhage
in a patent omphalomesenteric duct and risk
of malignancy [11].
We agre also with the importance of pathological study
of operative specimens due to possibility of presence of
ectopic gastric mucosa or pancreatic tissue in both Meckel’s
diverticulum and omphalomesenteric cyst; however, the presence
of gastric mucosa has not been reported in omphalomesenteric
fistula [5]. In our cases all specimens in H&E staining
did not reveal any abnormal tissue in the resected remnants.
We strongly underline, the need of careful clinical
examination of umbilical region due to neccesarity of exclusion
of abdominal wall defects. Omphalocele and umbilical
cord pseudocysts have been found to be associated with
aneuploidy when other fetal abnormalities are noted on prenatal
ultrasound scan [3]. Nobuhara recommend karyotype
determination in these specific cases. For these patients a widened
diagnostic evaluation protocol is proposed in cooperation
with clinical genetic consultant, for coexisting congenital
anomalies in other life-important organs [9].
It is important for the paediatric surgeons to have
a thorough understanding of the embryology, anatomy, presentation,
and relevant investigations for these anomalies. In
summary this knowledge results in a proper surgical management
and if needed, intraoperative modification of the surgical
technique in cases of complicated anomaly. This will
result, as in presented series, in an excellent outcomes of treated
patients.
10. Skandalakis JE, Gray SW (1994) In:
Embriology for surgeons: the embryological
basis for the treatment of congenital
anomalies, Williams and Wilkins,
Baltimore, pp 675–712
11. Storms P, Pexters J, Vandekerkhof J
(1988) Small omphalocele with ileal
prolapse through a patent omphalomesenteric
duct: a case report and review
of literature. Acta Chir Belg 88:
392–394
12. Ueno T, Hashimoto H, Yokoyama H, et
al (2003) Urachal anomalies: ultrasonography
and management. J Pediatr Surg
38: 1203–1207

Annals of Diagnostic Paediatric Pathology 2006, 10 (3–4): 137–140
© Copyright by Polish Paediatric Pathology Society Annals of
Merosin deficient congenital muscle dystrophy in children
– clinical features and retrospective immunohistochemical study
of own muscle biopsy material
Maciej Pronicki 1 , Hanna Mierzewska 2 , Tamara Szymañska-Dêbiñska 1 ,
Agnieszka Karkuciñska-Wiêckowska 1 , El¿bieta Karczmarewicz 3 , Tomasz Kmieæ 4
1 Department of Pathology
2 Division of Metabolic Disorders, Endocrinology and Diabetology
3 Department of Biochemistry and Experimental Medicine
4 Department of Neurology and Epileptology
The Children's Memorial Health Institute
Warsaw, Poland
Introduction
Abstract
Congenital muscle dystrophy (CMD) is a heterogenous group
of muscle disordes characterised by early clinical presentation,
usually at birth or in neonatal period. Dystrophic myopathic
pattern of skeletal muscle in biopsy consists a common
but non specific feature of all types. Typically muscle
shows extensive muscle fibre damage with intense interstitial
and replacement fibrosis. Clinically CMD is characterised
by weakness, hypotonia, atrophy of muscles, sometimes
joint contractures [8, 3].
Nearly half of CMD cases in humans is associated
with deficiency of laminin alpha 2 subunit (called merosin)
in muscle. Those cases are frequently associated with central
Address for correspondence
Congenital muscle dystrophy (CMD) is a heterogenous group of muscle disorders characterised by early
clinical presentation, dystrophic myopathic pattern of skeletal muscle in biopsy, weakness, hypotonia,
atrophy of muscles, sometimes joint contractures. Nearly half of CMD cases in humans is associated with
deficiency of laminin alpha 2 subunit (merosin). The gene LAMA2 responsible for the production of laminin
2 is located on chromosome 6q2. Pattern of inheritance is autosomal recessive. The most widely used
diagnostic procedure of merosin deficient CMD (MD CMD) is immunohistochemical confirmation in
skeletal muscle biopsy specimen. The aim of the study was retrospective immunohistochemical
examination of merosin in frozen archival skeletal muscle biopsy specimens. In available samples of 18
children selected for the study on the basis of age, skeletal muscle histology and clinical presentation, the
study revealed merosin deficiency in 4 children, two boys and two girls aged 4–16 months at the time of
biopsy. Authors analyse and discuss clinical presentation, muscle histology, spectrophotmetric analysis of
respiratory chain complexes in retrospectively revealed children with MD CMD.
Key words: congenital muscular dystrophy, merosin, merosin deficient congenital muscular dystrophy,
merosin immunohistochemistry
Maciej Pronicki, MD, PhD. Phone: +48 22 815 19 60
Department of Pathology Fax: +48 22 815 19 75
The Children’s Memorial Health Institute
Al. Dzieci Polskich 20
04-730 Warsaw, Poland
Diagnostic
Paediatric
Pathology
nervous system involvement with cerebral white matter lesions
which may be observed in MR imaging [12, 6]. Laminins
are proteins located in basement membranes of diverse
tissues. Merosin deficient CMD (MDCMD) is the first of
congenital dystrophies in which the biochemical defect and
genetic background has been identified. [11]. The gene
LAMA2 responsible for the production of laminin α2 is located
on chromosome 6q2 [4]. Inheritance is autosomal recessive.
Secondary merosin deficiency was also described in
a form of CMD with gene mutation localised on chromosome1q42
[2]. The degree of merosin reduction may differ in
individual patients what is reflected by different degree of
clinical severity of the disease [1, 9]. Some children may
even attain the ability to walk with or without support. In mo-

138
re severe cases myopathy is early and profound leading to death
due to hypoventilation and infection. As in other muscular
dystrophies there is still no specific cure for MDCMD but
physiotherapy and diverse supportive measures or even surgical
intervention may alleviate the symptoms and delay the
progression of the disease.
Since introduction of anti-merosin monoclonal antibodies
to laboratory practice, the diagnosis is mainly based
on immunohistochemical studies confirming the absence or
feduction of merosin in muscle or in skin. Skin biopsies may
reveal lack of merosin present in neural elements, but are not
suitable for detection of proteins involved in pathogenesis of
other myopathies so are not useful in differential diagnosis
in patients in whom the merosin is not lacking [7]. Some laboratories
perform genetic tests which ultimately proove the
diagnosis, but this method is not widely available. As is the
case for most other proteins studied immunohistochemically
in diagnosis of myopathies the normal expression of merosin
is sarcolemmal as shown on Fig. 1 [10].
Aim of the study
Our study consists of retrospective immunohistochemical
examination of merosin in frozen archival skeletal muscle
biopsy specimens obtained during the years 1996–2004,
when this method had not been used in routine biopsy assessment.
Selection of patients was based on clinical symptoms,
early presentation and dystrophic pattern of muscle damage
on light microscopy.
Patients and methods
Initially the group of 34 patients (15 boys and 19 girls) was
selected for the study from the whole pediatric biopsy material
(n=320) using the above mentioned criteria. Their age
ranged from 3 weeks to 6 years at the time of biopsy. Assessment
of archival muscle samples revealed that only 18 frozen
tissue blocks obtained from 9 girls and 9 boys are suitable
for further ivestigation due to technical reasons. Clinical
Fig. 1 Normal sarcolemmal expression of merosin in human skeletal muscle
shown immunohistochemically
presentation or suspicion at the time of biopsy comprised:
floppy child; spinal muscular atrophy; congenital dystrophy;
congenital myopathy; mitochondrial myopathy/respiratory
chain disorder. Archival histopathology reports concluded
the following: congenital muscle dystrophy, muscle dystrophy,
nonspecific myopathy, miositis.
Apart from controlled merosin immunohistochemistry
the muscle was reassessed in routine myopathology panel
of stains and reactions comprising: hematoxylin and
eosin; modified Gomori trichrome; oil red O; succinate dehydrogenase;
NADH dehydrogenase; cytochrome c oxidase;
acid phosphatase; myosin ATP-ase at pH 4,3/4,6/9,4. Spectrophotometric
assay of respiratory chain complexes was also
included into the study. Methods of biochemical investigation
were described earlier [5].
Results
Immunohistochemical analysis revealed 4 patients (two boys
and two girls) with merosin deficiency in muscle. Characteristics
of their clinical, pathological, and biochemical features
are as follows:
Patient 1
PR. A boy, born after uneventful pregnancy and delivery, as
a second child to unrelated parents. Birth weight was 2200 g,
length 48 cm, Apgar score was 8. From the beginning muscle
hypotonia and poor suckling were observed as well as hypomotility.
EMG was myopathic. Serum creatine kinase concentration
was markedly elevated – 809 – 1334 u/l. (normal
value – to 157 u/l.), and aminotranspherases were mildly elevated
(ASPAT – 63-95 IU, AlAT- 57-60 IU). During infancy
the child was floppy and his motor milestones were delayed.
He had slim habitus, dolichocephaly and long and slim fingers.
His muscles were atrophic and tendency to contractures were
observed. Brain MRI showed abnormal cerebral white matter
signal localised mainly in periventricular areas and in anterior
and posterior limbs of internal capsule (Fig. 2). Mild ventricular
enlargement and mild temporal atrophy were also found.
Muscle biopsy was performed at the age of 12 months.
Patient 2
WD. A boy born as a first child of unrelated parents. Pregnancy
and delivery was uneventful. Birth weight was 3180g. Apgar
was 8. After birth muscular weakness as well as mild cyanosis
were abserved. Congenital complex heart defect (ASD
+ VSD) was detected. The child was apathethic and hypokinetic.
His muscles were atrophic. The contractures of wrists,
knees and ankles developed as well as progressive funnel
chest deformity. Serum creatine kinase was markedly elevated
2348 – 1813 u/l. Aminotranspherases were normal. EMG
was myopathic. Muscle biopsy was performed at the age of
2,5 months. MRI of the brain was not performed.
Patient 3
NK. A girl born after first unevenful pregnancy and delivery
to noncosanguineous parents. Birth weight was 3090g,

Fig. 2 2 MRI lesions of patient 1
Apgar score was 10. From the 8th day of life she was markedly
hypotonic and had dyspnoe. X-ray examination showed
pneumonia and enlargment of the heart. EMG was myopathic.
ECG showed partial heart block (RBBB). Serum
creatine kinase concentration was markedly elevated – 2751
– 1780 u/l and aminotranspherases were slightly elevated
AlAT – 117 IU, AspAT – 117 IU. Muscle biopsy was performed
at the age of 4 months. Her motor milestones were
delayed but her intelectual development is very good at the
age of 2,5 years.
Patient 4
OE, a girl born as a first child to unrelated parents. Pregnancy
was complicated by maternal diabetes, delivery was uneventful.
Birth weigh was 2600g, Apgar score was 10. Since
neonatal period she was hypotonic. Progressive muscular
weakness was observed during physiotherapy. On physical
examination muscular wasting and lack of tendon reflexes
were detected. Her motor milestones was markedly delayed,
but intelect was spared. Creatine kinase concentration in serum
was increased – 894 – 1177 u/l. EMG was myopathic.
Brain MRI showed signal abnormality of hemispheric periventricular
white matter. U-fibers were also partially involved.
Lateral ventricles were mildly widened. Muscle biopsy
was performed at the age of 16 months.
Muscle biopsy –
histopathology and spectrophotometric assay
Muscle biopsy features of all children displayed the same
pattern concluded originally as muscle dystrophy or congenital
muscle dystrophy. Replacement fibrosis was most pronounced
in patient 4 in whom the biopsy was performed at
16 months. Apart from this finding there were no significant
differences in muscle lesions (Fig. 3 and 4). Immunohistochemical
examination revealed the same total lack of merosin
reactivity in all children (Fig. 5).
139
Fig. 3 Skeletal muscle histology of patient 2. Light microscopy of frozen
section stained with hematoxylin and eosin
Fig. 4 Skeletal muscle histology of patient 4. Light microscopy of frozen
section stained with hematoxylin and eosin
Fig. 5 Immunohistochemical reaction for merosin in MD CMD (patient 4).
Compare with Fig. 1

140
Spectrophotometric study was possible to start only in
patients 1 and 4 due to lack of muscle tissue in samples obtained
during biopsy, resultig from predominance of fibrous and
adipose tissue. However, in examined two muscle homogenate
samples, citrate synthase activity appeared below the level
of assay reliability for the assessment of respiratory chain complexes
activity (Patient 1. – 36 nmol/min/mg and patient 4. –
40 nmol/min/mg, reference value 96,5 – 150,1 nmol/min/mg).
Discussion
Clinical presentation of our MD CMD patients is characterised
by relative homogeneity. They were floppy children from
birth. Muscle hypotonia that was rather nonprogressive, was
observed permanently and motor milestones were markedly
delayed. All children had elevated concentration of creatine
kinase.
The above clinical picture is however non-specific
and it must be emphasised that there are no typical clinical
or biochemical features of MD CMD. All children had moderately
or markedly elevated creatine kinase concentration.
Increase of CK were in range of 1000 – 2700 u/l, what is rather
characteristic for all types of CMD. It is not so high as
in Duchenne muscular dystrophy where the level is over
10000 – 20 000 u/l. but is higher than in congenital myopathies
where the CK level is mildly elevated or normal. All
children had myopathic electromyography what is not characteristic
for myopathy or dystrophy.
References
1. Allamand V, Sunada Y, Salich MA, at
al (1997) Mild congenital muscular
dystrophy in two patients with an internally
deleted laminin alpha-2 chain.
Hum Mol Genet 6: 747–752
2. Brockington M, Sewry CA, Hermann
R, at al (2000) Assignment of a form of
congenital muscular dystrophy with secondary
merosin deficiency to chromosome
1q42. Am J Hum Genet 66:
428–435
3. Gordon E, Hoffman E, Pegoraro E
(2005) Congenital muscular dystrophy
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H, Chiannikulchai N, Guicheney P,
Grinal L (1994) Localisation of merosin
negative congenital muscular dystrophy
to chromosome 6q2 by homozygosity
mapping. Hum Mol Genet 3:
1657–1661
Typical pitfalls in clinical suspicion / interpretation
include differential diagnosis of so called “floppy infant syndrome”.
This syndrome is heterogenous and symptoms are
not characteristic so initial clinical suspitions were: spinal
muscular atrophy, congenital muscular myopathy, congenital
muscular atrophy, Pompe disease and other metabolic diseases,
as well as mitochondrial/OXPHOS disorders. MRI
findings may suggest other leukodystrophies especially of
metabolic origin (MRI of the first patient was described as
„metachromatic leucodystrophy or other metabolic leukodystrophy”).
All our patients showed relatively homogenous pattern
of skeletal muscle pathology in microscopic examination.
Histopathological examination showed dystrophic pattern
in routine stains which is not specific, so immunohistochemistry
with anti-merosin antibody is needed to confirm
the diagnosis.
In MD CMD it appeared difficult to obtain muscle
biopsy specimen containing representative fragment of skeletal
muscle tissue being suitable for biochemical investigation
of respiratory chain complexes activity in muscle homogenate.
Acknowledgement
5. Karczmarewicz E, Bielecka L, Kulczycka
H, Lorenc LS, Pronicka E
(1997) Analytical relibility of spectrophotometric
analysis of the activity of
mitochondrial respiratory chain complexes
in muscle homogenates. Diagn
Lab 33: 493–503
6. Lamer S, Carlier RY, Pinard JM (1998)
Congenital muscular dystrophy: use of
brain MR imaging findings to predict
merosin deficiency. Radiology 6:
811–816
7. Marbini A, Bellonoa MF, Ferrari A,
Lodesani M, Gemignani F (1997) Immunohistochemical
study of merosin-
-negative congenital muscular dystrophy:
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Acta Neuropathol 94: 103–108
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R, Trifiletti RR, Dobyns WB
(1995) Congenital muscular dystrophies:
clinical review and proposed
classification. Pediatr Neurol 13:
97–103
Research was supported by Internal Project of The Children’s
Memorial Health Institute Nr 100/05 (pricipal inverstigator
– M. Pronicki).
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V (1995) Clinical phenotype in
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