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HAL Id: tel-00862466 https://tel.archives-ouvertes.fr/tel-00862466 Submitted on 16 Sep 2013 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Développement du pancréas humain embryonnaire ex vivo / in vivo : La greffe musculaire : un nouveau modèle d’étude longitudinale et dynamique Carmen Capito To cite this version: Carmen Capito. Développement du pancréas humain embryonnaire ex vivo / in vivo : La greffe musculaire : un nouveau modèle d’étude longitudinale et dynamique. Médecine humaine et pathologie. Université René Descartes - Paris V, 2013. Français. <NNT : 2013PA05T015>. <tel-00862466>

Développement du pancréas humain embryonnaire ex vivo / in vivo

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Page 1: Développement du pancréas humain embryonnaire ex vivo / in vivo

HAL Id: tel-00862466https://tel.archives-ouvertes.fr/tel-00862466

Submitted on 16 Sep 2013

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Développement du pancréas humain embryonnaire exvivo / in vivo : La greffe musculaire : un nouveau

modèle d’étude longitudinale et dynamiqueCarmen Capito

To cite this version:Carmen Capito. Développement du pancréas humain embryonnaire ex vivo / in vivo : La greffemusculaire : un nouveau modèle d’étude longitudinale et dynamique. Médecine humaine et pathologie.Université René Descartes - Paris V, 2013. Français. <NNT : 2013PA05T015>. <tel-00862466>

Page 2: Développement du pancréas humain embryonnaire ex vivo / in vivo

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Page 4: Développement du pancréas humain embryonnaire ex vivo / in vivo

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A mon jury de thèse :

Je tiens en premier lieu à remercier Raphael Scharfmann, mon directeur de thèse :

tu m’as accueillie dans ton laboratoire il y a un peu plus de 4 ans. Après deux ans à

plein temps, j’ai dû reprendre mes activités cliniques. Concilier le clinicat et la thèse

n’a pas été chose facile mais tu as fait preuve de patience à mon égard et surtout tu

as su trouver les bonnes personnes pour m’aider à achever ce travail. J’ai beaucoup

appris à ton contact, notamment une certaine rigueur de raisonnement que j’espère

pouvoir retranscrire dans mon activité clinique mais aussi dans mon activité de

recherche à venir qui, je l’espère, passera par la poursuite d’une collaboration avec

ton équipe.

Au moment d’achever la première étape de mon parcours académique scientifique,

je tiens à remercier Monsieur Yves Aigrain, de m’avoir donné l’opportunité de

réaliser cette thèse, en me présentant à Raphael mais aussi en me libérant, autant

que possible, de mes activités cliniques ces deux dernières années. Je vous remercie

aussi d’avoir accepté de présider cette thèse. J’espère poursuivre cette activité de

recherche au sein de votre équipe médicale.

Je vous remercie Christian Pinset d’avoir pris sur votre temps pour rapporter cette

thèse et je suis fière de vous compter parmi les membres de mon jury.

Je tiens à remercier Dominique Berrebi d’avoir accepté de rapporter cette thèse :

chère Dominique, pendant mon passage à Robert Debré, j’ai eu la joie de travailler

avec toi au quotidien et de me rendre compte de la grande chance qu’avaient les

équipes chirurgicales de cet hôpital de te compter à leur côté.

Page 5: Développement du pancréas humain embryonnaire ex vivo / in vivo

Je tiens à remercier Roberto Mallone d’avoir accepter d’examiner ce travail. Vos

travaux sur l’immunopathologie du diabète et sa génétique me rendent fière de vous

compter dans mon jury de thèse.

Madame Delezoïde, vous ne vous souvenez certainement pas mais, encore jeune

étudiante de médecine, j’attendais vos cours d’embryologie avec impatience et, en y

assistant, découvrait la « magie » de cette spécialité. Si certains enseignants sont

capables de transmettre leur passion pour leur spécialité, vous comptez certainement

parmi ceux là et, pour moi, aux toutes premières places. J’ai découvert et pris goût à

l’embryologie avec vous et je vous redis encore la fierté que j’ai de vous compter

dans les membres de mon jury.

Aux collègues du laboratoire :

Je tiens en premier à remercier Marie Thérèse Gage Soufflot : chère MT, si ce

surnom d’agent secret que je t’ai donnée te va si bien (en tout cas je trouve !!!) c’est

que tu es capable, en un temps record, d’extraire des données majeures d’un fouillis

de manip générées par une thésarde dans le rush tous les lundi….. Je me dois d’une

idée initiale et le reste je te le dois. Je tiens vraiment à t’exprimer ici toute ma

reconnaissance (éternelle….) pour l’aide apportée pour l’analyse de ce nouveau

modèle.

Virginie Aiello Lorenzo : tu m’as fait faire mes premiers pas à l’animalerie des

SCID, tu m’as appris que même si ce n’était que des souris il fallait respecter

certaines règles, tu m’as montré comment greffer sous la capsule rénale (si fine !!!!)

les pancréas fœtaux. Tu m’as accompagnée ensuite pour les greffes musculaires, tu

m’as montré comment disséquer des E12 de souris (si petits pancréas !!!!), nous

avons tâtonné ensemble sur les moyens de greffer et surtout retrouver… ces E12

dans le quadriceps des souris. Ce projet a démarré avec toi et je te dois beaucoup

beaucoup beaucoup…. De plus, tu as toujours été une oreille attentive à mes excès

d’enthousiasme scientifique en sachant les modérer par ton expérience et les pousser

quand j’en avais besoin. Raphael a de la chance de te compter dans son équipe et

j’ai de la chance de te compter parmi mes amies…..

Page 6: Développement du pancréas humain embryonnaire ex vivo / in vivo

Séverine Pechberty, ma voisine puis mon amie de paillasse, un puits de

connaissance tant théorique que pratique. Je ne pensais pas qu’il était nécessaire de

comprendre en détail les moindres étapes d’une manip avant de t’entendre

m’expliquer les choses. Tu as écouté mes angoisses, mes craintes pendant 4 ans. Tu

as aussi partagé mes joies au labo, en France et ailleurs… Raphael a de la chance

de te compter dans son équipe, même si ces derniers temps nous ne sommes plus sur

le même site…Miss you…

Je tiens aussi à remercier les autres membres du laboratoire avec lesquels j’ai

toujours eu beaucoup de plaisir à me retrouver et qui m’ont, chacun à leur niveau,

apporté beaucoup, et particulièrement Latif, scientifique passionné et à qui je

souhaite toute la réussite qu’il mérite ; Olivier, ta connaissance scientifique n’a

d’égal que ta qualité d’enseignant et j’espère vraiment avoir la possibilité un jour de

collaborer avec toi sur toutes ces molécules fluorescentes et tous ces virus aux noms

quelquefois obscurs que tu maîtrises si bien ; Bertrand, ton flegme apparent est

inversement proportionnel à tes qualités scientifiques ; Estelle, nous avons beaucoup

échangé sur l’Hôpital et les difficultés de concilier la Recherche et la Clinique, je te

souhaite beaucoup de joies dans tes nouvelles fonctions nantaises ; Fanny, l’année a

été difficile pour toutes les deux, plus que quelques semaines avant que « i wish you

nothing but the best… » ; Olivia, Mylène, Kanetee, Djalee, Vikash, Ghislaine,

Samia, Masaya, Andrea, Kathleen, Elodie, Aurore.

Aux collègues hospitaliers :

Monsieur Alaa Elghoneimi, merci de m’avoir donné l’opportunité de poursuivre ce

travail pendant mon clinicat dans votre service et merci encore à vous et à votre

brillante équipe pour votre enseignement.

Madame Sabine Sarnacki, j’ai réalisé mon master 2 sous votre direction il y a

maintenant quelques années, sur un sujet fort interessant et qui, je l’espère, profitera

à de nombreuses petites patientes. Je vous remercie de votre confiance d’alors et

j’espère que nous continuerons à échanger à l’interface de la Science et de la

Clinique.

Page 7: Développement du pancréas humain embryonnaire ex vivo / in vivo

Monsieur Christophe Chardot, ces presque deux années à tes côtés au « theater »

comme disent les anglais m’ont permis d’avancer tant sur le plan clinique que

scientifique. Cette exigence dont tu fais preuve vis-à-vis de toi et des patients est une

qualité qui m’a certainement servi à structurer ce travail au moment où il en avait

besoin.

A mes cochefs pendant ces trois dernières années : Béné, Elodie et Audrey pour

m’avoir aidée pour l’organisation de mon temps scientifique. Audrey tu as été

particulièrement précieuse en ces derniers instants. Je suis ton obligée jusqu'à la fin

de mes jours.

A mes internes, sans la compétence desquels je n’aurais pas pu arriver au bout de ce

travail. Je pense particulièrement à Liza, Benoit, Jérome, Sara, Aias aka « et

voilà ! », Erik aka young Padawan, Maddalena aka fireball, Lara aka Lara Kroft.

A mes rencontres scientifiques :

Au moment où j’achève l’écriture de cette thèse je ne peux m’empecher de penser à

vous Mesdames Nadine Binart et Luisa Dandolo. Si j’ai un jour décidé de

poursuivre une aventure scientifique, c’est parce que lors de mon Master 2 j’ai pu

bénéficier de votre enseignement. Ce dernier m’a rendu les méandres de la Science

moins obscurs mais surtout m’a fait prendre conscience, par la passion que vous

mettez à l’enseigner, qu’il n’est finalement de limite que l’imagination dans votre

discipline. Je vous dois tant….

A mes amies de toujours :

Julie, Pascale, Karine, je n’ai pas été très disponible pour nous ces dernières années

mais vous avez toujours été là pour partager mes doutes mais aussi mes joies…, la

vie quoi…. Il faut dire que entre la côte est africaine et la côte est américaine, vous

m’avez laissée bien seule dans l’ancien monde……Julie, merci de ton aide pour

finaliser ce travail et cela, surtout sous le soleil d’Hawaï, je sais ce que cela t’a

coûté… Moi, perso, j’aurais refusé….

Page 8: Développement du pancréas humain embryonnaire ex vivo / in vivo

A mes parents :

Sans vous, vos conseils, vos encouragements incessants, votre parcours qui est un

exemple à mes yeux, je ne serais jamais allée aussi loin….. Akewani …. Maman,

sans ton aide précieuse pendant les congés de maternité et en ces derniers instants je

n’aurai certainement pas pu finir ce travail.

Page 9: Développement du pancréas humain embryonnaire ex vivo / in vivo

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Page 10: Développement du pancréas humain embryonnaire ex vivo / in vivo

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Page 12: Développement du pancréas humain embryonnaire ex vivo / in vivo

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Chronologie du développement du pancréas............................................................ - 14 - Embryologie pancréatique humaine ....................................................................................... - 14 - Embryologie pancréatique murine (la souris)......................................................................... - 23 -

Embryogenèse moléculaire endocrine : les données murines comparativement aux

données humaines ....................................................................................................... - 25 - Les facteurs de transcription clés de la morphogenèse pancréatique précoce ....................... - 26 - Les facteurs de transcription clés de la différenciation du lignage endocrine ........................ - 33 - Les facteurs solubles............................................................................................................... - 36 -

Les modèles d’étude du développement pancréatique humain .............................. - 39 -

Rationnel du travail de thèse...................................................................................... - 44 -

������ ��������������������������������������������������������������������������������������������������������������������� ��!"��

Article : Le muscle représente un site de greffe permissif pour le développement du

pancréas immature humain ....................................................................................... - 48 - 1. Stratégie et méthode...................................................................................................... - 48 - 2. Résumé des résultats ..................................................................................................... - 50 -

B����������#��$#��%#���&#� �������������������������������������������������������������������������������������� "'

Le choix du site musculaire .............................................................................................80

Les potentielles applications cliniques de la greffe intramusculaire ...........................81

Discussion des résultats obtenus et des questions soulevées par ces résultats ...........83

Le transfert de gènes et les applications potentielles pour l’étude du développement pancréatique .....................................................................................................................85

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The mouse muscle as an ectopic permissive site for human pancreatic

development

Carmen Capito1, Marie-Thérèse Simon

1, Virginie Aiello

1, Anne Clark

2, Yves

Aigrain3 Philippe Ravassard

4, Raphael Scharfmann

1

1 INSERM U845, Research Center Growth and Signalling, Université Paris

Descartes, Faculté de Médecine Cochin, Paris, France

2 Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and

Metabolism, Churchill Hospital, Oxford UK

3 Necker Enfants Malades University hospital, Université Paris Descartes, Paris,

France

4 Université Pierre et Marie Curie-Paris 6, Biotechnology and Biotherapy Team,

Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière (CRICM),

UMRS 975, CNRS, UMR 7225, INSERM, U975, Paris, France.

En cours de revue au journal : Diabetes

Abstract

Whilst sporadic human genetic studies

have permitted some comparisons between

rodent and human pancreatic development,

the lack of a robust experimental system

has not permitted detailed examination of

human pancreatic development. We

previously developed a xenograft model of

human fetal pancreas grafted under the

kidney capsule of immune-incompetent

mice, which allowed the development of

human pancreatic beta cells. Here, we

compared development of human fetal

pancreatic grafts at a less complicated and

efficient alternative site under skeletal

muscle epimysium with renal capsule

grafts and with murine muscle or renal

capsule grafts. We demonstrated that

human pancreatic beta cell development

occurs more slowly (weeks) than murine

pancreas (days) both by differentiation of

pancreatic progenitors and by proliferation

of developing beta cells. The superficial

location of the skeletal muscle graft and its

easier access permitted in vivo lentivirus-

mediated gene transfer with a GFP labeled

construct under control of the insulin or

elastase gene promoter which targeted beta

cells and non-endocrine cells respectively.

This model of engraftment under the

skeletal muscle epimysium is a new

approach for longitudinal studies, which

allows localized manipulation to determine

the regulation of human pancreatic

development.

Introduction

Understanding the mechanisms that

control human pancreatic islet

development remains a target for

deciphering the pathophysiological

mechanisms of disease and for developing

innovative therapeutic approaches. In

rodents, genomic gene disruption

experiments have enabled inference of the

transcriptional regulatory network and

paracrine soluble factors that control

pancreas specification and later endocrine

and exocrine fate determinations. In

humans, rare genetic deficiencies are

currently the way that regulatory factors

for pancreatic islet development are

determined.

In rodents, the pancreas-committed

endodermal region of the foregut first

expresses the transcription factor

pancreatic and duodenal homeobox 1

(PDX1); mice and humans deficient in

PDX1 lack a pancreatic gland (1,2). PDX1

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later becomes restricted to mature

pancreatic delta cells and beta cells where

it activates insulin gene expression (3).

These undifferentiated PDX1+ pancreatic

progenitors proliferate through the

mesenchymal effector FGF10 (4) and

differentiate into endocrine, ductal and

acinar cells. The endocrine specification

rests upon the transient expression of the

basic helix loop helix factor, neurogenin3

(NGN3), and NGN3-deficient mice lack

pancreatic endocrine cells (5). NGN3-

expressing cells are unipotent endocrine

precursors (6) and differentiate into the

four pancreatic endocrine cell types (alpha,

beta, delta and PP cells, producing

respectively glucagon, insulin,

somatostatin and pancreatic polypeptide).

Subsequently, combinations of additional

transcription factors will determine the

specific fate and stability of each

pancreatic endocrine cell type (7). As an

example, NKX2.2 is first expressed in

early mouse pancreatic PDX1+

progenitors. Afterwards it is maintained in

early endocrine progenitors along with

NGN3 (7,8) and later becomes restricted to

endocrine cells (except delta cells) as islets

develop (9). In rodent pancreas, NKX2.2

plays a major role in maintaining beta cell

identity (10).

Less is known on pancreatic

development in human. This is at least in

part due to the difficulty in accessing

properly staged human fetal pancreatic

tissues and to the frequently poor quality of

such tissues. This paucity of information is

also due to the absence of dynamic assays

to follow human beta cell development

from pancreatic progenitors, and to the

lack of assays to genetically modify

pancreatic cells at different stage of their

development. Many aspects seem

conserved between rodent and human

pancreatic development. For example,

different arguments derived from human

genetic studies strongly suggest that PDX1

and NGN3 play similar roles during rodent

and human pancreatic development

(2,11,12). But differences seem to exist on

the pancreatic role of other transcription

factors such as for example MAFB or

GATA family members (13,14). This is

not unexpected since, while rodent and

human adult pancreatic beta cells share a

large number of similarities, a number of

data also indicate marked differences

between species (15,16).

We previously developed and

validated a model of xenograft of human

fetal pancreas under the kidney capsule of

immune-incompetent SCID mice. We

demonstrated that this grafting model was

permissive for proper development of the

fetal tissue into a functional human

endocrine pancreas (17). However,

grafting under the kidney capsule is time-

consuming, limiting the number of SCID

mice to be grafted and the amount of data

to be produced. Moreover, due to its deep

anatomical localization, in close contact to

the kidney parenchyma following grafting,

the growing pancreas is difficult to

genetically modify to examine lineage

tracing or longitudinal transcription factor

expression.

In this context, we have developed an

alternative site by grafting human fetal

pancreas under the epimysium of the thigh

muscle. Using this model we have

examined in detail the major steps and

transcription factor expression that take

place during human pancreatic

development. Finally, we have

demonstrated the feasibility of cell-type,

specific virus-mediated gene transfer to

allow lineage tracing to observe islet and

non-endocrine cell development in human

fetal pancreas.

Research Design and Methods

Mouse and human pancreases Pregnant Swiss mice were purchased from

the Janvier breeding center (Janvier, Le

Genest, France). At 12.5 days of gestation

(E12.5), mice were killed by CO2

asphyxiation, according to the guidelines

of the French Animal Care Committee and

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the embryos were harvested. The dorsal

pancreas was isolated and preserved in

Hank’s balanced salt solution until use.

Human fetal pancreases were extracted

from tissue fragments (18) that were

obtained immediately after elective

termination of pregnancy between 7 and 9

weeks of gestation, in compliance with the

French bioethics legislation. Approval was

obtained from the Agence de Biomedecine,

the French competent authority along with

maternal written consent.

Animals and grafting into SCID mice Severe combined immunodeficiency

(SCID) mice (Charles River, L’arbresle,

France) were kept in isolators supplied

with sterile-filtered, temperature-controlled

air. Cages, bedding and drinking water

were autoclaved. Food was sterilized by X-

ray irradiation. The French animal ethics

committee approved these studies. For

grafting, 6- to 8-week-old SCID mice were

anesthetized with a ketamine/xylazine mix.

Fetal pancreases were implanted (1

pancreas per graft), using a dissecting

microscope, either under the kidney

capsule as previously described (17) or

under the muscle epimysium of either the

biceps femoris or the gluteus superficialis.

At different time points following grafting,

grafts were removed, fixed in formalin

3.7% and embedded in paraffin.

Immunolabelling and quantification

Graft sections (4-5 µm-thick) were

prepared and processed as previously

described (19). The following primary

antibodies were used for immunostaining:

mouse anti-BrdU (Amersham,

Courtaboeuf, France); rabbit anti-

carboxypeptidase A (Biogenesis Ltd,

Poole, UK, 1/600); rabbit anti-GFP

(Abcam, Paris, France, 1/1,000); mouse

anti-glucagon (Sigma, St Louis, MO, USA,

1/2,000); mouse anti-insulin (Sigma,

1/1000); mouse anti-Ki67 (BD, Franklin

Lakes, NJ, USA 1/20); sheep anti- human

NGN3 (R&D systems, Lille, France,

1/400); mouse anti- mouse NGN3 (BCBC,

1/1,000); mouse anti-NKX2.2

(Developmental Studies Hybridoma Bank,

1/50); rabbit anti-pan-cytokeratin (Dako,

Trappes, France 1/500); rabbit anti-PDX1

(1/1,000) (20); mouse anti-somatostatin

(BCBC, 1/500). The secondary antibodies

were: dyelight or FITC conjugated anti-

rabbit antibodies (Beckman Coulter,

Villepinte, France, 1/200); texas red or

FITC anti-mouse antibodies (Beckman

Coulter, 1/200); alexa fluor anti-rabbit

antibodies (Biogenex, Fremont, CA, USA,

1/400). For NGN3, revelation was

performed using the Vectastain ABC kit

(Vector, Malakoff, France).

Cell proliferation For cell proliferation analyses, mice were

injected with bromodeoxyuridine (BrdU,

50 mg/kg) 4 h before being killed. To

measure the proliferation of PDX1+

pancreatic progenitors, we counted the

frequency of BrdU+ nuclei among PDX1+

cells. To measure the proliferation of beta

cells, we counted the frequency of BrdU+

nuclei among insulin+ cells. At least 1,000

cells per graft were counted in each

condition. The same tissues were also

stained with Ki67 antibody and

quantification performed as described

above, as a second readout for cell

proliferation.

Electron microscopy For standard electron microscopy, human

pancreatic grafts were removed, cut into

1mm cubes and fixed overnight in 2.5%

glutaraldehyde in 0.1M phosphate buffer

pH 7.2. For gold immunolabelling the

fixative solution was 2.5%

paraformaldehyde plus 0.5%

glutaraldehyde in 0.1M phosphate buffer

pH 7.2. All fixed specimens were stored at

4°C until processed into resin; Spurr’s

resin (Elektron Technology UK, Stansted

UK), was used for structural observations

and London Resin Gold (LRG) (Elektron

Technology UK) was used for

immunolabelling. Ultrathin (70nm) section

were cut onto nickel grids, contrasted with

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uranyl acetate and lead citrate and viewed

with a Joel 1010 electron microscope.

LRG sections were immunogold labeled

for insulin using guinea-pig anti-insulin

(Sigma, UK) and protein A gold (British

Biocell International, Cardiff UK)

Induction of diabetes with alloxan To determine the capacity of the human

graft to regulate the glycaemia of the

mouse, grafted (3 months grafts) and non-

grafted (control mice) SCID mice were

injected intravenously with alloxan

(Sigma-Aldrich, 90 mg/kg body weight),

which is known to destroy rodent, but not

human, beta cells (21).Glucose

concentrations were measured on blood

collected from the tail vein, once a week

during 4 weeks, using a portable glucose

meter (OneTouch Vita, Lifescan France,

Issy les Moulineaux, France). To confirm

the contribution of the graft to the

normalization of blood glucose values in

the host, grafts were removed 30 days after

the injection of alloxan and blood glucose

concentrations were measured during one

more week. Circulating insulin

concentrations were measured at day 0

after alloxan injection by Elisa method

(Mercodia insulin Elisa, Human, Mercodia

AB, Uppsala, Sweden).

Lentivirus-mediated gene transfer The lentiviral construct pTRIP

deltaU3.RIP405-GFP has been previously

described (22). The lentiviral construct

pTRIP deltaU3.ElastaseP-GFP was derived

from the pTRIP deltaU3.RIP405-GFP.

Briefly, the insulin promoter was removed

by MluI and BamHI digestion and replaced

by a 500 bp fragment of the Elastase

promoter flanked with the same MluI and

BamHI restriction sites. The Elastase PCR

fragment was amplified using phusion high

fidelity polymerase (Finnzyme) from

Elastase GFP pcDNA3 plasmid kindly

provided by David Tosh (University of

Bath, UK) using forward primer 5’

ACGCGTCAGATCAGCTTATCGTATG

AA 3’ and reverse primer 5’

GGATCCCGAGACCACTGCCCCTTGC

3’. Lentiviral vectors were prepared and

titrated as described (22). Grafted (3-

month grafts) SCID mice were

anesthetized. Hundred micro liters of the

lentiviral vector solutions (105 transduction

units) were injected using 32 gauge

needles into multiple sites of the

developing human pancreas. Pancreases

were harvested at 7, 14 and 30 days

following injections and used for

immunofluorescence analyses.

Results

Development of mouse and human fetal

pancreas upon grafting under the

muscle epimysium or under the kidney

capsule The renal capsule has been widely used as

a grafting site for mature rodent and human

islets and for fetal pancreas (17,23,24). We

first compared the development of mouse

fetal pancreas grafted either under the

kidney capsule (renal graft) or at the level

of the muscle epimysium (muscular graft).

We found that undifferentiated E12.5

pancreases developed efficiently and in a

similar fashion at both sites. Two weeks

following grafting, insulin- and glucagon-

expressing cells were present, forming

islet-like structures with insulin-positive

clusters surrounded by glucagon-

expressing cells (Fig. 1A, D). Insulin-

positive cells expressed PDX1 (Fig. 1B,

E). Duct-like structures also developed

similarly at both sites (Fig. 1A-F).

Interestingly, acinar cells, visualized either

following carboxypeptidase-A staining

(Fig. 1C, F) or following amylase staining

(data not shown), developed at none of the

grafting sites.

We next compared human fetal pancreas

development following grafting under the

muscle epimysium or the kidney capsule.

Three months following grafting, insulin-,

glucagon- and somatostatin-expressing

cells were observed on both sites (Fig. 2A,

B, D, E). Beta cells stained positive for

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PDX1 (Fig. 2C, F) and were never found

positive for glucagon (Fig. 2A, D),

supporting the differentiated status of such

newly formed beta cells (25,26). Ductular

structures that stained positive for pan-

cytokeratin were observed on both sites

(Fig. 2G, J). Acinar cells that stained

positive for carboxypeptidase-A developed

from human fetal pancreas grafted on

either site (Fig. 2G, H, J, K). This contrasts

with the findings with grafted mouse

pancreas (Fig. 1C, F).

We then characterized in more detail beta

cells that developed from human fetal

pancreases grafted under the muscle

epimysium. For functional analysis, three

months following grafting, we destroyed

endogenous mouse beta cells with alloxan

(21), which gave rise to increased

glycaemia in non-grafted mice (Fig. S1).

Among five grafted mice, one did not

regulate its glycaemia, had low circulating

human insulin levels and poor beta cell

differentiation (Fig. S1). In four mice,

glycaemia was efficiently regulated with

circulating human insulin levels reaching

0.6ng/ml and efficient beta cell

differentiation (Fig. S1). Of note, all four

mice became hyperglycemic upon after

removal of the grafts.

For morphological analysis, electron

microscopy of the human fetal graft was

performed 8 months after grafting. It

indicated the presence of well-granulated

alpha and beta cells and a developed

capillary network (Fig. 3A). Immunogold-

electron microscopy indicated the presence

of insulin within structurally mature beta-

cell granules (Fig. 3B,C).

These data demonstrated that pancreatic

development of mouse or human pancreas

was similar upon grafting under the muscle

or the kidney capsule. Since grafting at the

muscle site was technically easier, faster

and could potentially allow perturbation

experiments, we thus performed muscular

grafting in the remaining part of this study.

PDX1+ pancreatic progenitors following

grafting

We analyzed the presence of PDX1+

pancreatic progenitors following grafting.

In engrafted mouse fetal pancreas, all

PDX1+ cells stained positive for insulin

two weeks following grafting (Fig. 1E).

This indicated that within two weeks, the

pool of PDX1+/endocrine- negative,

pancreatic progenitors had been depleted.

In contrast, many PDX1+/INS- negative

cells were observed 3 months following

grafting of human fetal pancreas (Fig. 2F).

We next measured the proliferation of

PDX1+ pancreatic progenitors at different

time points following grafting of human

fetal pancreas. Two weeks after grafting,

16% of PDX1+ cells stained positive for

Ki67 and 12% of PDX+ cells stained

positive for BrdU following a 4h pulse

(Fig. 4). These proportions decreased by

nearly two fold 13 weeks following

grafting, and further decreased 60 weeks

following grafting (Fig. 4).

Endocrine progenitors following

grafting We analyzed the expression of NGN3, a

transient marker of endocrine progenitors

(5) following grafting. Two weeks

following grafting of mouse fetal pancreas,

NGN3+ endocrine progenitors were not

observed (Fig. 5A). In grafted human

pancreas, NGN3 was detected at weeks 2,

8, 13 and 19 post-grafting, its expression

being undetectable at weeks 37 and 60

following grafting (Fig. 5B). Thus, NGN3

is expressed during at least 19 weeks in the

grafted human fetal pancreas.

During mouse pancreatic development,

NKX2.2 is expressed in early pancreatic

progenitors and later becomes restricted to

endocrine cells (except delta cells) as islets

develop (9). In grafted human fetal

pancreas, two weeks following grafting,

NKX2.2 was expressed in the first

developing endocrine cells, and also in

many endocrine-negative cells (Fig. 6).

Many NKX2.2+/endocrine- cells remained

present till at least week 19 following

grafting. At later post-grafting time points,

the number of differentiated endocrine

Page 59: Développement du pancréas humain embryonnaire ex vivo / in vivo

��EG���

cells increased and such cells stained

positive for NKX2.2. In parallel, the

number of NKX2.2+/endocrine-cells

decreased (Fig. 6).

Taken together, such results indicated that

in mouse, the pool of pancreatic and

endocrine progenitors was depleted within

a two week period whereas in human

pancreas, pancreatic and endocrine

progenitors remained present for at least

four months following grafting, creating a

pool of cells giving rise to endocrine cells.

Beta cell proliferation in grafted

pancreases Two weeks following grafting, 8.9% beta

cells that developed from mouse fetal

pancreas incorporated BrdU after a 4h

BrdU pulse and 16.15% beta cells stained

positive for Ki67 (Fig. 7A). In grafted

human fetal pancreas, beta cell

development increased during a 60-week

time period with the development of larger

islet-like structures (Fig. S2). Thirteen

weeks following grafting, 2% beta cells

incorporated BrdU following a 4h pulse

and 3.5% beta cells stained positive for

Ki67 (Fig. 7B). These proportions

measured in grafted human fetal

pancreases were lower than the ones

measured in grafted mouse pancreas and

further decreased by more than 5 fold 60

weeks following grafting (Fig. 7B).

Virus-mediated gene transfer into the

developing human pancreatic cells We finally asked whether it was feasible to

specifically target human pancreatic cell

types in the above-described model of

human fetal pancreas that has developed

under the muscle epimysium. Three

months following grafting of human fetal

pancreases under the muscle epimysium,

we injected within the developing tissue

lentiviral vectors that expressed GFP under

the control of either the insulin promoter or

the elastase promoter. Grafts were

removed and analyzed 7-30 days later.

When GFP was under the control of the

insulin promoter, we labeled insulin+ cells

with this approach (Fig. 8), while

endocrine-negative cells that could

represent nascent acinar cells were labeled

when GFP was under the control of the

elastase promoter (Fig. S3).

Discussion

During the past years, major progress has

been made on understanding factor

controlling mouse pancreatic islet-cell

development. This is at least in part due to

the development of a large number of

innovative approaches, for example

transgenic mice for gain- and loss-of-

function experiments or in vitro assays to

screen for signals that modulate pancreatic

development (27). On the other hand,

information on human pancreatic islet

development remained scarce. A first

major limitation to efficient progress on

human pancreas development is the

inability to longitudinally track or impact

on human pancreas development with

models developed so far. A second

limitation is the difficult access to human

fetal pancreases corresponding to specific

stages of development in large-enough

quantity and quality.

In this work, we developed and validated a

new grafting model for study of human

pancreatic islet development. Moreover,

this model permitted to perform virus-

mediated gene transfer in specific human

pancreatic cell types.

We previously developed a model of

xenograft of human fetal pancreas under

the kidney capsule of SCID mice. This

model was permissive for pancreatic

development and gave rise to functional

human beta cells (17). However, grafting

under the kidney capsule is technically

challenging when performing a large

number of grafts, and time-consuming.

Moreover, our attempts to perform virus-

mediated gene transfer into the developing

human pancreas grafted under the kidney

capsule failed due to difficulties to access

Page 60: Développement du pancréas humain embryonnaire ex vivo / in vivo

��E4���

the graft and hemodynamic drastic

variations when trying to mobilize the

kidney (data not shown). In this context,

we modified our initial model and used the

muscular site as an alternative. The

muscular site has been used in the past to

graft mature islets. For example the

forearm muscle was used with success in

humans for islet auto-transplantation after

total pancreatectomy for severe hereditary

pancreatitis (28). In addition, a murine

model of islets grafted within the cremaster

muscle was developed and showed an

efficient intra-islet blood supply after two

weeks of engraftment (29). Moreover, islet

injections into biceps femoralis in the rat or

into gracilis muscle in minipig induced a

sustained reversal of diabetes (30,31).

Finally, human islets grafted into the

muscle of the forearm could be imaged,

demonstrating their survival 1 year after

following implantation (32). On the other

hand and to the best of our knowledge, the

muscle has not been used as a grafting site

for undifferentiated fetal pancreas and it

was unknown whether this site was

permissive for proper pancreatic cell

differentiation. Here, we have

demonstrated that rodent and human fetal

pancreas properly develop upon grafting

under the muscle epimysium. This site

could thus represent a new site for grafting

of human pancreatic progenitors. Indeed,

protocols are now available to generate

PDX1+ pancreatic progenitors from hESCs

(33). These progenitors need to be grafted

into mice to differentiate into functional

pancreatic endocrine cells (34). There is

however a recurrent discussion on the best

site for grafting of either mature islets or

pancreatic progenitors that would develop

into functional beta cells (35). In this

context, the muscular epimysium

represents an intersting site as: i)

functional beta cells develop from human

fetal pancreatic progenitors; ii) the

developing graft can be easily removed if

necessary for oncological reasons; iii) it

should be possible to image the developing

beta cells, using approaches similar to the

ones used for imaging beta cells grafted in

muscle forearm (32).

In this model of muscular grafting, within

two weeks following transplantation of a

E12.5 mouse pancreas, differentiated cells

developed, while the pool of PDX1+

pancreatic progenitors was depleted, and

all PDX1+ cells expressed insulin. This

result perfectly fits with rodent data

obtained either in vivo or in vitro (19,36).

In contrast, three months following

grafting of human fetal pancreas, many

PDX1+/Insulin- cells were still observed.

They were efficiently proliferating, based

on both Ki67 staining and BrdU

incorporation. These proliferation indices

decreased at later time points post-grafting.

Taken together, such results suggest that

pancreatic cell differentiation remains

active during a long period in humans. It is

however important to keep into account

that in humans, the presence of

PDX1+/Insulin-negative cells represents an

indirect sign of active differentiation as

PDX1 remains expressed within pancreatic

duct cells during adult life (37). But more

direct arguments indicate that endocrine

cell differentiation takes place during a

long period following grafting of human

fetal pancreas. Two weeks following

grafting of mouse E12.5 pancreas,

endocrine progenitors that expressed the

transient NGN3 marker could not be

observed as is the case in vivo and in in

vitro rodent models (5,19,36). On the other

hand, NGN3-positive cells could be

observed within the grafted human fetal

pancreas up to at least 19 weeks following

engraftment. NGN3 expression was

undetectable 37 and 60 weeks following

grafting. This suggests that the process of

endocrine differentiation occurs over

periods of weeks and is probably finished

at the end of pregnancy in humans. Similar

results were obtained by looking at the

expression of NKX2.2. In rodents,

NKX2.2 is expressed in early pancreatic

progenitors. Its expression is later

maintained in mature endocrine cells (9).

Page 61: Développement du pancréas humain embryonnaire ex vivo / in vivo

��FH���

In the grafted human fetal pancreas, cells

positive for NKX2.2 but negative for

endocrine markers could be detected for at

least 19 weeks following grafting.

Based on this longitudinal study of PDX1,

NGN3 and NKX2.2 expression patterns in

the grafted human fetal pancreas, we

propose that endocrine cell differentiation

takes place during a long time window

during development. This long time-frame

in humans should be useful to attempt to

capture and amplify in vitro human

pancreatic progenitors as performed in the

case of neural progenitors (38). Moreover,

as previously suggested (14) such long

lasting developmental windows of human

pancreatic development could explain why

haploinsuffficiency of a number of

transcription factors such as GATA6,

HNF1A, HNF1B, and HNF4 cause

diabetes in humans and not in rodents.

Beta cell proliferation in this grafting

model reproduced beta cell proliferation

observed in vivo both for rodent and

human pancreas. This is the case for mouse

E12.5 pancreases engrafted during two

weeks, where 8.9% of beta cells

incorporated BrdU as reported for newborn

mouse pancreas (4). This is also the case

for human fetal pancreases where 2% beta

cells incorporated BrdU 3 months

following grafting with 3.5% beta cells

positive for Ki67. Such levels decreased

one year following grafting with 0.2% beta

cells that incorporated BrdU and 0.7% that

stained positive for Ki67. These data

obtained with Ki67 antibodies are in

accordance to those reported in fetal and

neonatal pancreases at the same

developmental stages (39,40). Of note, the

above-described dynamic model of human

pancreatic development permitted to

further support data obtained with Ki67

antibodies with data obtained following

BrdU injections. To our knowledge, such a

type of experiments on human PDX1+

progenitors or human fetal beta cells

entering the S-phase using BrdU

incorporation had not been described in the

past due to the lack of proper experimental

system. Such a dynamic model of beta cell

proliferation should thus be useful to better

dissect the mechanisms that regulate

human beta cell proliferation during the

perinatal period (41).

Overall, our results demonstrated the

efficiency of the muscular fetal pancreatic

graft model to recapitulate pancreatic

development. The difference observed

between mouse and human pancreatic islet

development after grafting further

emphasizes the specificity of each species

regarding cell differentiation, maturation

and proliferation. The fact that we

successfully performed cell type specific in

vivo lentiviral-mediated gene transfer in

human pancreas grafted under the muscle

epimysium represents a first step to the use

of this model in a dynamic manner for

gain- and loss-of-function experiments.

Author contribution R.S. is guarantor and takes full

responsibility for the manuscript and its

originality. C.C. conceived experiments,

researched data, contributed to the

discussion, and wrote the manuscript. M.-

T.S, V.A. and researched data. A.C

performed the electron microscopy. P.R.

contributed tools. R.S. conceived

experiments, contributed to the discussion

and also wrote the manuscript.

Acknowledgments C.C. was supported by the Fondation pour

la Recherche Médicale and by the

Association des Jeunes Diabétiques. This

work was supported by grants to RS from

the Beta Cell Biology Consortium (grant

1U01DK089571-01), from the 7th

Framework Program of the European

Union under grant agreements n° 241883,

from the Innovative Medicine Initiative

under grant agreement n° 115005, from the

bilateral program Bundesministerium fur

Bildung und Forschung (BMBF) ANR,

convention number 2009 GENO10502 and

Page 62: Développement du pancréas humain embryonnaire ex vivo / in vivo

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from the Laboratoire d’Excellence

consortium Revive

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!CBE� ,E�E� �����0ED� 9�A�O� �A�O� �A�O� �A7:A� In order to further test the ability of the graft to

respond to acute hyperglycemia, we performed a glucose tolerance test following a 16-hour

fast. Four mice grafted with human fetal pancreas developed during 8 months were submitted

to a high dose of glucose by intraperitoneal injection 9�� !'X'� ��D�� ,EC'��: ��D� ����D�

'��B��E�!E����ED�7H=�FH=���D���H�!C���E���-�E�� ��E� C�BEB�C��A�#�!��� insulin� �EB�E�C���

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C�BEB�C��A� One mouse (1.2) was hyperglycemic (up to 300ng/dl) 30 minutes after injection and

this correlated with undetectable human insulin secretion and poor beta cell differentiation. In

three mice, glycemia was regulated thanks to an efficient human insulin secretion (from 4 to

10ng/ml) five minutes after glucose injection�

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Page 101: Développement du pancréas humain embryonnaire ex vivo / in vivo

Value of 18F–fluoro-L-dopa PET

in the Preoperative Localization

of Focal Lesions in Congenital

Hyperinsulinism1

Carmen Capito, MD

Naziha Khen-Dunlop, MD

Maria-Joao Ribeiro, MD, PhD

Francis Brunelle, MD

Yves Aigrain, MD

Celia Cretolle, MD, PhD

Francis Jaubert, MD

Pascale De Lonlay, MD, PhD

Claire Nihoul-Fekete, MD

Purpose: To retrospectively compare fluorine 18 (18F) fluoro-L-dopapositron emission tomography (PET) and pancreatic ve-nous sampling (PVS) in the preoperative differentiation ofdiffuse from focal congenital hyperinsulinism (CHI) andlocalization of focal lesions.

Materials and

Methods:

This study was approved by the institutional ethical com-mittee, and informed consent for the research study wasobtained from the parents of all subjects. Fifty-one pa-tients evaluated for focal CHI between January 1, 1995,and January 31, 2008, were included. Thirty five under-went PVS evaluation alone, and 16 underwent a PET eval-uation alone. The sensitivity values of each technique forthe diagnosis and localization of focal lesions were com-pared in regard to results of surgery and pathologic analy-ses. In each patient, perioperative treatment was re-viewed, and the presence of postoperative hypoglycemiawas assessed as evidence of incomplete resection. Com-parisons of the sensitivity values and recurrence rateswere performed by using the Fisher exact test in regard tothe number of patients. Comparisons of median age,weight, or number of biopsies were performed with atwo-tailed unpaired Mann-Whitney U test. A differencewith P , .05 was considered significant.

Results: For PVS and PET groups, there was no error in differentiat-ing focal from diffuse forms. PVS was not completed in fourof 35 patients. In 27 (87%) of 31 patients in whom PVS wascompleted and 13 (81%) of 16 patients in whom PET wascompleted, preoperative localization of the focal lesion was inaccordance with the surgical findings (P 5 .7). Although notsignificant, the number of biopsies performed before discov-ering the focal lesion was higher in the PET group comparedwith the PVS group (P 5 .06). Inadequate localization oc-curred in two (6%) patients in the PVS group and five (31%)patients in the PET group at initial preoperative imagingstudy; these patients underwent repeat surgery for residualCHI (P 5 .03).

Conclusion: 18F–fluoro-L-dopa PET is equivalent to PVS in the charac-terization of CHI but does not provide localization of thelesion as precisely as does PVS.

r RSNA, 2009

1 From the Departments of Pediatric Surgery (C. Capito,

N.K., Y.A., C. Cretolle, C.N.), Radiology (F.B.), Pathology

(F.J.), and Pediatrics (P.D.L.), Necker-Enfants Malades

Hospital, AP-HP, 149 rue de Sevres 75015 Paris, France;

and Biomedical Imaging Institute, Life Sciences Division

CEA, Frederic Joliot Hospital, Orsay, France (M.J.R.). Re-

ceived August 18, 2008; revision requested October 25;

final revision received February 26, 2009; accepted

March 17; final version accepted June 1. Address corre-

spondence to C. Capito (e-mail: carmendoc@hotmail

.com).

R RSNA, 2009

OR

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216 radiology.rsna.org ▪ Radiology: Volume 253: Number 1—October 2009

Note: This copy is for your personal, non-commercial use only. To order presentation-ready copies for

distribution to your colleagues or clients, use the Radiology Reprints form at the end of this article.

Page 102: Développement du pancréas humain embryonnaire ex vivo / in vivo

Congenital hyperinsulinism (CHI) isthe major cause of pediatric postna-tal persistent hypoglycemia and oc-

curs with an estimated incidence of one in30 000 to one in 50 000 live births innorthern countries (Europe, Canada,United States) (1). This abnormality ischaracterized by inappropriate insulinrelease, and early diagnosis and treat-ment are required to prevent severeneurologic damage (2). Histopathologi-cally, two major forms have been de-scribed: diffuse and focal (3,4). The dif-fuse form is characterized by dysregula-tion of insulin secretion that involves allthe pancreatic b cells. Treatment is gen-erally medical, but in rare cases whenthis treatment fails, near-total pancrea-tectomy is required (5). Pancreatec-tomy, the only procedure that can pre-vent hypoglycemia, results in iatrogenicdiabetes mellitus as a consequence ofthe procedure (6). In the focal form,insulin secretion dysregulation is local-ized to one region of the pancreas (7,8),and children with the focal form can becured by using complete resection of thefocal lesion (9,10). In cases of residualdisease, postoperative recurrence of hy-poglycemia will occur.

The clinical presentations of the twoforms are indistinguishable. Consequently,after correction of hypoglycemia and main-tenance of normal glucose levels, the ma-jor challenges in the treatment of childrenwith this abnormality are to characterizethe histopathologic form and, in childrenwith the focal form, to localize the lesionprior to surgery. Until 2002, pancreaticvenous sampling (PVS) by using catheter-ization of the portal system was themethod of choice for the diagnosis of thefocal form. By producing a detailed vascu-lar pancreatic map of the insulin concen-tration in each region of the pancreas,

this imaging study allows accurate diagno-sis and precise anatomic localization offocal lesions (11). However, it is invasiveand technically difficult to perform (12).Positron emission tomography (PET)with fluorine 18 (18F) fluoro-L-dopa wasintroduced in Necker-Enfants MaladesHospital (Paris, France) in 2002. Thisfunctional imaging study requires only pe-ripheral venous access and does not re-quire the patient to be hypoglycemic atthe time of the study. The study is basedon the capability of neuroendocrine cellsto take up L-DOPA and convert it to dopa-mine by decarboxylation (13). Research-ers reported that 18F–fluoro-L-dopa PEThas been shown to aid in the accuratedifferentiation of focal from diffuse forms(14,15), but the precision of the anatomiclocalization is limited by the spatial reso-lution of the nuclear technique itself(16,17). The purpose of our study was toretrospectively compare 18F–fluoro-L-dopaPET and PVS for the preoperative differ-entiation of diffuse from focal CHI and forlocalization of focal lesions.

Materials and Methods

Patients

The institutional ethics committee ofNecker-Enfants Malades Hospital ap-proved this retrospective study, and in-formed consent was obtained from theparents for clinical and research studies.

We retrospectively reviewed the surgicaland pathologic results in 51 patients whowere suspected of having and weretreated for focal CHI between January 1,1995, and January 31, 2008. These pa-tients were suspected of having a preop-erative diagnosis of focal CHI, in view ofthe resistance to medical treatment andthe findings at PVS or PET, which sug-gested a focal lesion. In all patients, sur-gical and pathologic results served as thereference standard for the diagnosis ofeither the diffuse or the focal form and fordetermination of the exact location of thelesion. Thirty-five patients underwentPVS evaluation between January 1, 1995,and March 31, 2002, and 16 children un-derwent an 18F–fluoro-L-dopa PET evalu-ation between November 1, 2002, andJanuary 31, 2008. Sixteen male patientsand 19 female patients underwent PVS,whereas 13 male patients and three fe-male patients were examined by usingPET. These differences in sex were notsignificant. The median age at diagnosis ofCHI was 1 day, with an age range of1–270 days for the PVS group and an agerange of 1–150 days for the PET group,and the difference between the groupswas not substantial. Respective medianage and weight at the time of radiologicevaluation were 5 months and 6.7 kgin the PVS group and 3 months and 7.1 kgin the PET group. The differences be-tween the groups were not significant(age, P 5 .06; weight, P 5 .8).

Published online before print

10.1148/radiol.2532081445

Radiology 2009; 253:216–222

Abbreviations:

CHI 5 congenital hyperinsulinism

PVS 5 pancreatic venous sampling

ROI 5 region of interest

SUV 5 standardized uptake value

Author contributions:

Guarantors of integrity of entire study, C. Capito, N.K., C.

Cretolle, P.D.L., C.N.; study concepts/study design or data

acquisition or data analysis/interpretation, all authors;

manuscript drafting or manuscript revision for important

intellectual content, all authors; manuscript final version

approval, all authors; literature research, C. Capito,

M.J.R., F.B., Y.A., F.J.; clinical studies, all authors; statis-

tical analysis, C. Capito, M.J.R., Y.A.; and manuscript

editing, C. Capito, N.K., M.J.R., F.B., Y.A., C.N.

Authors stated no financial relationship to disclose.

Advance in Knowledge

n The results of this study suggestthat albeit 18F–fluoro-L-dopa PETis an excellent examination for thecharacterization of the morpho-logic type of congenital hyperinsu-linism (focal vs diffuse), it fails toallow precise localization of thelesion for the surgeon.

Implications for Patient Care

n Children with focal forms of hy-perinsulinism can be cured withcomplete resection of the focallesion.

n The limitations of 18F–fluoro-L-dopa PET examination must beconsidered when planning surgeryto avoid unnecessary extendedpancreatic resections and to pre-vent long-term pancreatic insuffi-ciency.

n Improvements in spatial resolu-tion of the PET signal and the useof hybrid machines, such as PET/CT, may be helpful in obtainingbetter anatomic localization, per-mitting organ-sparing surgery.

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PVS Studies

The PVS studies (12) were performed byone author (F.B., with 10 years of experi-ence), who was the initiator of the tech-nique in Necker-Enfants Malades Hospi-tal. All medical treatments were stopped5 days before the examination. With ad-ministration of a general anesthetic, thepatients were maintained in a hypoglyce-mic state, with a venous blood glucoselevel between 2 and 3 mmol/L, and retro-grade transhepatic catheterization of theportal system was performed. At least 10venous blood samples from throughoutthe pancreatic venous network were col-lected for measurements of plasma glu-cose, insulin, and C-peptide levels. Dur-ing phlebography, a pancreatic venousmap of insulin levels was obtained (Fig 1).This map allowed the localization of thefocal lesion by using a simultaneous com-parison of the insulin–C-peptide ratiosand glucose levels at different anatomiclocations. A focal lesion was character-ized by an increase in insulin–C-peptideratios in one or two contiguous locations,when low levels of insulin and C-peptidewere obtained in the remaining gland. Indiffuse forms, insulin and C-peptide levelswere increased throughout the gland. Theratio of C-peptide to insulin was calculatedin each sample. Near a focal lesion, thisratio was about 1.0 (meaning that the levelsfor secretion of C-peptide and insulin by theb cells were the same), and it increased asthe distance from the lesion increased.

18F–fluoro-L-dopa PET Studies

The 18F–fluoro-L-dopa PET studies (18)were performed by an author (M.J.R.,with 7 years of experience), who was theinitiator of the technique in Necker-En-fants Malades Hospital. Intravenous injec-tion of 18F–fluoro-L-dopa (mean dose ofradioactivity injected, 4.2 MBq per kilo-gram of body weight 6 1.0 [standard de-viation]) was performed, and a thoraco-abdominal PET scan (three-dimensionalacquisition mode) was begun between 45and 65 minutes after radiotracer injec-tion. Depending on the height of the child,data were acquired in two to four bedpositions. The data sets were correctedfor scatter by using a model-based correc-tion, allowing simulation of a map of sin-gle scatter events. The images were re-

constructed by using an attenuation-weighted ordered subset expectationmaximization iterative algorithm, withfour iterations and six subsets. The finalspatial resolution in reconstructed imageswas approximately 6 mm. Three regionsof interest (ROIs) were identified in theright, middle, and left areas of the pan-creas; these areas corresponded to thehead (right side of the spine) (Fig 2), body(in front of the spine) (Fig 3), and tail (leftside of the spine) (Fig 4), respectively.The sizes of these ROIs varied from onechild to another; that is why the spine wasthe major anatomic landmark for inter-preting the location of the lesion. Themean radioactivity concentration in eachROI (in becquerels per milliliter), mea-sured at 60 minutes after injection, wasdivided by the injected dose of the radio-tracer (in becquerels) and was correctedfor the radioactive decay between injec-tion and scan start times and body weight(in grams) to calculate standardized up-take values (SUVs). Next, values for theratio between the SUV of the region

where the lesion is located and the meanpancreatic SUV were calculated. In dif-fuse forms, the values for the ratio be-tween the SUV of the region where thelesion is located and the mean pancreaticSUV are about 1.0. In contrast, in focalforms, the values for the the ratio be-tween the SUV of the region where thelesion is located and the mean pancreaticSUV are between 1.2 and 2.0. We consid-ered that an abnormal focal 18F–fluoro-L-dopa uptake for the pancreatic regionshould be reflected by a ratio between theSUV of each of the three ROIs and themean pancreatic SUV to be greater than1.2. The radiotracer has an early biliaryexcretion and late urinary elimination(gallbladder, liver, followed by kidneysand bladder, high signals during the ex-amination).

Surgery

In cases of radiologically confirmed focalCHI, partial elective pancreatectomywas recommended. Meticulous inspec-tion of the anterior wall of the pancreas

Figure 1

Figure 1: Vascular pancreatic map shows insulin concentrations in 3-month-old male infant with focal

CHI. Low levels of insulin were observed in all but one point of splenic vein (circled box with numbers). This

location was associated with hypersecretion of insulin (serum insulin level, 407 mIU/L [2827 pmol/L]), si-

multaneous hypoglycemia (serum glucose level, 1.9 mmol/L), and insulin–C-peptide ratio of 1.4. Results of

this examination were highly suggestive of focal lesion in distal body of pancreas. Values in each box repre-

sent serum glucose level (in millimoles per liter), serum insulin level (in milli–international units per liter),

and insulin–C-peptide ratio, from top to bottom. To convert values for serum insulin level to Systeme Interna-

tional units (in picomoles per liter), multiply by 6.945.

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was performed, with a search for an obvi-ous lesion in the area suggested by thefindings at the preoperative radiologic ex-amination (head, body, or tail). Three bi-opsy specimens were taken in the threemorphologic areas of the pancreas forpathologic confirmation of the focal formof the disease (endocrine cells with abun-dant cytoplasm and large irregular nucleiin the pathologic zone and resting b cellswith small nuclei outside the focal lesion).Analyses were performed by one author(F.J., with 20 years of experience). If nolesion was seen on the anterior wall, thepancreas was mobilized from tail to headand inspected on the posterior wall. Ifthere was still no obvious lesion, multiplebiopsies were performed, first in the areasuggested by findings at imaging and then,if needed, at different points throughoutthe gland. For each biopsy, a contempo-raneous pathologic examination was per-formed until the focal lesion was found.After resection of the lesion, specimenswere examined from an adequate marginof normal tissue. The surgical procedurewas always performed by the same team(C.N. and Y.A., with 20 years of experi-ence in CHI surgery).

Evaluation

The surgical treatment (number of biop-sies performed before finding the focallesion and the size of the pancreas re-sected during surgery) and postopera-tive outcomes in the group of patientswho underwent preoperative PVS werecompared with postoperative outcomesin the group of patients who underwentpreoperative PET for evaluation. Pan-creatic resections for focal lesions var-ied from resection of a single well-delin-eated lesion to resections of one or mul-

tiple pancreatic areas. A resection wasjudged as complete if there was no post-operative recurrence of hypoglycemia.A resection was judged as incomplete ifthere was recurrence of hypoglycemia.In cases of incomplete resection, a sec-ond radiologic examination (the sameas initially performed) was performedto locate the persisting focus.

Statistical Analysis

Quantitative data were expressed withmedians and ranges. Diagnostic sensi-tivity of each examination was definedas the ratio of the number of patients inwhom a diagnosis of the focal form wasdetermined by using PVS or PET and inwhom the diagnosis was confirmed by us-ing surgery and pathologic analysis to thetotal number of patients in each group(PVS or PET). Accuracy or sensitivity forlocalization was defined as the ratio of thenumber of patients with correct localiza-tion of the lesion by using PVS or PETcompared with correct surgical localiza-tion to the total number of patients ineach group (PVS or PET). Comparisonsof sensitivity values and recurrence rateswere performed by using the Fisher exacttest, regarding the number of patients.Comparisons of median age, weight, ornumber of biopsies were performed witha two-tailed unpaired Mann-Whitney U

test. A difference with a P value of lessthan .05 was considered significant. Thestatistical analysis was performed withsoftware (StatView for Windows, version5.0; SAS Institute, Cary, NC).

Results

Diagnostic Sensitivity: Focal versus

Diffuse Form

PVS was successfully performed in 31patients. In four other patients, the ex-amination was not completed becauseof difficulties in maintaining the patientsin a stable hypoglycemic state during theexamination or because of an inability toperform transhepatic pancreatic cathe-terization. These four patients were, then,excluded from the rest of the analysis. PETexaminations were successfully completedin all patients examined, uneventfully.

Thus, as summarized in Table 1, the

Figure 2

Figure 2: Transverse 18F–fluoro-L-dopa PET

image in 6-month-old male infant shows focal lesion

(arrow) in head of pancreas. Diagnosis was con-

firmed at surgery. Focal lesion corresponds to large

uptake region localized in front of and on right side of

spine. Large posterior regions of increased uptake

correspond to kidneys. Despite poor spatial resolu-

tion of images, lesion was accurately localized by

using PET and was removed completely.

Figure 3

Figure 3: Transverse 18F–fluoro-L-dopa PET

image in 4-month-old male infant shows focal

lesion (arrow) in body of pancreas, localized in

front of spine.

Figure 4

Figure 4: Transverse 18F–fluoro-L-dopa PET

image in 5-month-old male infant shows focal

lesion (arrow) in tail of pancreas (far from spine,

on left side of spine).

Table 1

Diagnostic Sensitivity of PVS and PET

for Focal CHI

Clinical Data

PVS

Group

PET

Group

No. of patients 35 16

No. with diagnosis of focal

form 31 16

No. with procedure failure 4 0

No. with erroneous diagnosis 0 0

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diagnostic sensitivity for distinguishing be-tween the focal and the diffuse forms wasexcellent (100%) for both PVS and PETgroups. There was no error in differentiat-ing the focal form from the diffuse form.

Accuracy of Approximate Localization of

Focal Lesions

Because precise localization was notpossible with PET imaging alone, thesensitivity values of the preoperative ra-diologic localization with PVS and PETwere compared for the three major pan-creatic areas (head, body, tail).

During surgery, macroscopic focal le-sions were seen in 24 (77%) of 31 pa-tients in the PVS group and in 11 (69%) of16 patients in the PET group. In 27 (87%)of 31 patients in the PVS group and in 13(81%) of 16 patients in the PET group,preoperative localization of the lesion wasperformed, in accordance with the surgi-cal findings. In 74% (35 of 47) of ourpatients, the lesion was visualized at sur-gery, and this factor helped in the perfor-mance of the resection, and the use of thefindings from pathologic analysis was offurther assistance. The radiologic and

surgical features of the four patients inthe PVS group in whom misdiagnoses oflocalization occurred are summarized inTable 2. The three cases of misdiagnosisin the PET group are described in Figures5–7. There was no significant differencebetween the two groups in the accuracyof localization of focal lesions (P 5 .7).

The median number of biopsies weperformed before we discovered thefocal lesion was higher in the PETgroup (median number, six; range,1–12) than it was in the PVS group(median number, three; range, 0–12),

Figures 5–7

Figures 5: Coronal 18F–fluoro-L-dopa PET image shows inaccurate localization of focal lesion in 2-month-old female infant. Results suggested focal form (FF) le-

sion in body of pancreas (arrow). Perioperative findings indicated bifocal form with one lesion in inferior part of head of pancreas and another in wall of descending duo-

denum. Repeat surgery was performed because of incomplete resection of this bifocal lesion initially.

Figure 6: Coronal 18F–fluoro-L-dopa PET image shows inaccurate localization of focal form (FF) lesion in 2-month-old male infant. Results suggested focal lesion in

body of pancreas (red arrow). Gallbladder signal (green arrow) was well identified. Lesion was not identified during surgical procedure, and body of pancreas was re-

sected, as pointed out by using PET. After persistent hypoglycemia was noted postoperatively, this patient underwent repeat surgery, and focal lesion in head of pancreas

was resected.

Figure 7: Coronal 18F–fluoro-L-dopa PET image shows inaccurate localization of focal form (FF) lesion (red arrow) in 5-month-old male infant. Results suggested

focal lesion in body of pancreas. Common bile duct (CBD) and right ureter (green arrows) were well identified because they were abnormally dilated. After persistent hy-

poglycemia was noted postoperatively, this patient underwent repeat surgery, and focal lesion in head of pancreas was resected.

Table 2

Misdiagnoses at PVS

Patient No./Sex/Age (mo) Pancreatic Location at PVS Pancreatic Location at Perioperative Localization Extent of Resection

1/M/5 Head Body (lesion seen) Elective resection; patient was cured

2/F/7 Head Body (lesion seen) Elective resection; patient was cured

3/F/4 Body Tail (lesion seen) Elective resection; patient was cured

4/F/7 Body Lesion not seen and not found perioperatively; recurrence,

with localization in head at postoperative PVS

Resection of body and tail and secondary near-total

pancreatectomy; patient was cured

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although the difference between thegroups was not significant (P 5 .06).

In the PVS group, two (6%) patientsunderwent further surgery for residualCHI, which was diagnosed after recur-rence of hypoglycemia. In one case, thisrecurrence resulted from incomplete re-section of the lesion, although findingsfrom an intraoperative pathologic exami-nation suggested free margins; the resec-tion was completed during a second pro-cedure. In the other case, partial resec-tion was performed, without successfulpreoperative localization of the lesion (pa-tient 4, Table 2); a second procedure wasperformed successfully.

In the group examined by usingPET, five (31%) patients underwent fur-ther surgery for residual CHI, whichwas diagnosed after recurrence of hypo-glycemia. In three of these patients, thefirst resection did not include the entirelesion, although the intraoperative patho-logic examination results suggested freemargins. The resection was completedwith a second procedure. In two otherpatients, the lesion was not found duringthe initial surgical procedure, when resec-tion of the region identified by using PETwas performed. After early recurrence ofhypoglycemia, these two patients under-went a second PET examination, whichconfirmed the persistence of focal dis-ease. The resection was successfully com-pleted during a second surgical proce-dure.

Of the 47 children in this study, 40(85%) were cured after the initial surgi-cal procedure, However the reexplora-tion rate was significantly higher in thePET group (P 5 .03).

Discussion

18F–fluoro-L-dopa PET is now the imagingmethod of choice for the examination ofpatients with CHI (13–15). Unlike PVS,PET evaluation can be performed easily,does not require patients to be anesthe-tized or hypoglycemic during the exami-nation, and does not require suspensionof medical treatment prior to examina-tion. Moreover, the radiation dose islower (1 mSv/kg for PVS vs 0.4 mSv/kgfor PET), and there is no associated riskof deep venous thrombosis. The only lim-

itation of this technique is that the radio-tracer is not available in all PET centers.

If one considers the approximate lo-calization of the focal lesion, with the pan-creas divided into three main areas, theresults of PET and PVS were comparable(81% and 87%, respectively, of lesionscorrectly identified). This finding con-firms that 18F–fluoro-L-dopa PET is notonly an accurate noninvasive techniquefor diagnosing focal forms of CHI but alsoa good examination for initial localizationof the pancreatic focal lesion (17). Theaim of surgery is to completely removethe lesion, while sparing as much of thepancreas as possible to prevent long-termpancreatic insufficiency. However, whenwe compared PET with PVS, we foundthat the lack of precision in the localiza-tion of the lesion by using PET can haveadverse consequences for both surgeonand patient: In this study, the repeat sur-gery rate was five times higher in the PETgroup.

Preoperative visualization of these fo-cal lesions is difficult because of theirsmall size (mean length, 10 mm) (17).Identification of these focal lesions is alsodifficult because their texture may beidentical to that of the surrounding nor-mal gland and because they occur eithersuperficially or deep within the pancreaticgland and may not be readily visible. In74% (35 of 47) of our patients, the lesionwas visualized at surgery, and this factorhelped in the performance of the resec-tion, and the use of the findings frompathologic analysis was of further assis-tance. For patients in whom the lesioncould not be seen macroscopically duringsurgery, the results of preoperative radio-logic studies and intraoperative histologicstudies were essential in guiding the re-section.

As with all functional imaging, 18F–fluoro-L-dopa PET demonstrates a bio-logical activity, and in this case, it is theactivity of hyperactive b cells in the pan-creas. The signal seen is proportional tothe activity rather than to the size of thelesion (13). Moreover, precise anatomiclandmarks, usually used in conventionalimaging, are not apparent (19,20). Whenwe compared 18F–fluoro-L-dopa PETwith PVS, the major limitation of PETwas its limited spatial resolution. Be-

cause PVS can be used to screen theentire pancreatic venous network, it al-lows precise anatomic localization of thefocal lesion. Although the difference interms of accuracy of approximate local-ization (P 5 .7) between the two tech-niques was not significant (probably be-cause of insufficient power, given thesmall number of patients), we found adifference, although not significant (P 5

.06), in the number of biopsies that hadto be performed during surgery. Thisdifference may be the consequence ofgreater difficulties in precisely locatingthe lesion with PET, despite the appar-ent comparable rate of “approximate”localization by using the three pancre-atic areas (head, body, and tail). Thisstudy had several shortcomings. Be-cause of its retrospective nature and thesmall number of patients in each group,this study failed to display significantdifferences between PVS and PETgroups concerning the accurate localiza-tion of focal lesions. Nevertheless, therepeat surgery rate in the patients in thePET group was significantly higher, sug-gesting better outcomes in patientstreated with PVS. This finding is favor-able in regard to the use of hybrid ma-chines, such as PET/computed tomog-raphy, which will permit better ana-tomic localization and more precisedelineation of focal CHI (19–22).

In conclusion, 18F–fluoro-L-dopa PETis an excellent way to differentiate focalfrom diffuse CHI, but this techniquefails to give precise localization of thelesion, partly because of the absence offixed anatomic landmarks (such as thecommon bile duct, splenic and mesen-teric blood vessels, spleen, and duode-num).

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Morphological Mosaicism of the Pancreatic Islets:

A Novel Anatomopathological Form of Persistent

Hyperinsulinemic Hypoglycemia of Infancy

C. Sempoux, C. Capito, C. Bellanne-Chantelot, V. Verkarre, P. de Lonlay,Y. Aigrain, C. Fekete, Y. Guiot, and J. Rahier

Department of Pathology (C.S., Y.G., J.R.), Cliniques Universitaires Saint Luc, 1200 Brussels, Belgium;

Departments of Pediatric Surgery (C.C., Y.A., C.F.) and Pathology (V.V.), Unite 845 Institut National de la

Sante et de la Recherche Medicale (C.C.), and Reference Center of Metabolic Diseases (P.d.L.) Assistance

Publique-Hopitaux de Paris (AP-HP) Hopital Necker-Enfants Malades, Universite Paris Descartes, Faculte

de Medecine, 75743 Paris, France; and Department of Genetics (C.B.-C.), AP-HP Groupe Hospitalier

Pitie-Salpetriere, Universite Pierre et Marie Curie, 75651 Paris, France

Background: Morphological studies of the pancreas in persistent hyperinsulinemic hypoglycemia

of infancy (PHHI) have focused on the diagnosis of focal vs. diffuse forms, a distinction that de-

termines the optimal surgical management. ABCC8 or KCNJ11 genomic mutations are present in

most of them.

Aim: Our aim was to report a new form of PHHI with peculiar morphological and clinical

characteristics.

Research Design and Methods: Histopathological review of 217 pancreatic PHHI specimens re-

vealed 16 cases morphologically different from diffuse and focal forms. They were analyzed by

conventional microscopy, quantitative morphometry, immunohistochemistry, and in situ

hybridization.

Results: Their morphological peculiarity was the coexistence of two types of islet: large islets with

cytoplasm-rich b-cells and occasional enlarged nuclei and shrunken islets with b-cells exhibiting

little cytoplasm and small nuclei. In small islets, b-cells had abundant insulin content but limited

amount of Golgi proinsulin. Large islets had low insulin storage and high proinsulin production and

were mostly confined to a few lobules. No evidence for KATP channels involvement or 11p15

deletion was found. Genomic mutations for ABCC8, KCNJ11, and GCK were absent. Patients had

normal birth weight and late hypoglycemia onset and improved with diazoxide. Ten were cured

by limited pancreatectomy. Six recurred after surgery and were medically controlled.

Conclusion: This new form of PHHI is characterized by a morphological mosaicism. Pathologists

should recognize this mosaicism on intraoperative frozen sections because it is often curable by

partial pancreatectomy. The currently unknown genetic background does not involve the classical

genomic mutations responsible for diffuse and focal PHHI. (J Clin Endocrinol Metab 96: 0000–0000,

2011)

Persistent hyperinsulinemic hypoglycemia of infancy

(PHHI), a syndrome that was first recognized by

McQuarrie (1), has been repeatedly attributed to nesidi-

oblastosis, a particular histological feature that is character-

ized by the presence of numerous small clusters of endocrine

cells in the vicinity of the pancreatic ducts (2). Nesidioblas-

tosis was considered a permanent b-cell proliferation, lead-

ing to an increased b-cell mass that was responsible for

ISSN Print 0021-972X ISSN Online 1945-7197

Printed in U.S.A.

Copyright © 2011 by The Endocrine Society

doi: 10.1210/jc.2010-3032 Received December 27, 2010. Accepted August 25, 2011.

Abbreviations: H&E, Hematoxylin and eosin; ISH, in situ hybridization; LOH, loss of

heterozygosity; PET, positron emission tomography; PHHI, persistent hyperinsulinemic hy-

poglycemia of infancy; PVS, pancreatic venous sampling; SUR1, sulfonylurea receptor 1.

O R I G I N A L A R T I C L E

E n d o c r i n e R e s e a r c h

J Clin Endocrinol Metab, December 2011, 96(12):0000–0000 jcem.endojournals.org 1

J Clin Endocrin Metab. First published ahead of print September 28, 2011 as doi:10.1210/jc.2010-3032

Copyright (C) 2011 by The Endocrine Society

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hyperinsulinism. However, the proliferation index and the

fractional volume of b-cells are not higher in the pancreas

of hypoglycemic infants than in controls, and nesidioblasto-

sis is not specific to the disease in that it is also observed in the

pancreas of normoglycemic neonates and infants (3, 4).

Since the 1990s, histological retrospective analyses of

case series led several groups to characterize two forms of

PHHI: the focal and the diffuse forms (3–7). In the first

form, a small focal endocrine lesion, often invisible to the

naked eye, is responsible for insulin hypersecretion. This

lesion is an adenomatous hyperplasia made from the con-

fluence of histologically roughly normal islets with few

non-b-cells at the periphery of a b-cell central core. Ab-

normal, large b-cell nuclei are often observed within the

lesion but are not seen outside of the focal lesion where the

islets exhibit small nuclei and less cytoplasm, with evidence

of low proinsulin synthesis. This focal form is curable by a

partial pancreatectomy restricted to the lesion. The second

form is diffuse and involves the whole pancreas. Its histolog-

icalhallmark is thepresenceofabnormally large b-cell nuclei

scatteredthroughoutmost islets (3–6). Inbothforms, infants

have a high birth weight, hypoglycemia occurs in the neo-

natalperiod,anddiazoxide isnearlyalways ineffective (8,9).

Neither clinical nor biological data can be used to differen-

tiate the focal from the diffuse forms. To distinguish the two

forms, portal venous catheterization with selective venous

sampling for insulin measurements (10) and more recently

noninvasive imaging using positron emission tomography

(PET) with [18F]fluoro-L-DOPA (18F-PET) are useful (11,

12). Intraoperative frozen sections are essential, not only to

confirm the diagnosis made based on imaging but also to

determine whether the suspected area effectively corre-

sponds to a focal lesion and is completely resected (13).

The genetic cause of many cases has been identified. In

most diffuse forms, hyperinsulinism is due to inactivating

mutations in ABCC8 or KCNJ11, which are located on

chromosome 11p15 and code for the sulfonylurea recep-

tor 1 (SUR1) and Kir6.2 proteins, respectively, the two

subunits of ATP-sensitive K (KATP) channels, resulting

in abnormal channels (14, 15). In the focal forms, in ad-

dition to a paternally inherited inactivating mutation in

ABCC8 or KCNJ11, there is a loss of heterozygosity in the

11p15 region, leading to the occurrence of focal adeno-

matous hyperplasia because of an imbalance between im-

printed genes involved in cell proliferation (H19 and

IGF2) and a loss of the antiproliferative factor p57 (16–

20). Infrequent cases of congenital hyperinsulinism unre-

lated to KATP channels have also been reported; most of

these cases were sensitive to diazoxide. These cases may be

due to dominant activating mutations of the GLUD1

gene, which codes for glutamate dehydrogenase (21, 22),

or due to activating mutations of GCK, which codes for glu-

cokinase (23, 24); these are two enzymes that are involved in

the metabolic control of insulin secretion. Loss-of-function

mutations in the HADH gene and mutations in HNF4a

can also produce infantile hyperinsulinism (25–29). The

rarity of these forms, which are unrelated to defects of the

KATP channels, explains why exhaustive descriptions of

their pathological characteristics are limited.

Among the 217 cases of operated PHHI that we col-

lected over 25 yr, 16 cases (7.4%) did not fit with the usual

types. They displayed peculiar pancreatic morphologies

and had a distinct clinical presentation, with normal birth

weight in the majority of the cases, a late onset of hyper-

insulinism, and a relative sensitivity to diazoxide. Fur-

thermore, when genetic analyses were performed in

these infants (n 5 13), genomic mutations of ABCC8,

KCNJ11, and GCK were ruled out, and no similar peculiar

pathologies were found in cases with similar mutations. The

aim of the present study is to report the histological charac-

teristicsof thesepatients topermit intraoperative recognition

and diagnosis of this particular form of PHHI that can often

be cured by a limited pancreatectomy.

Materials and Methods

On the basis of their specific histological features alone, we identi-

fied 16 cases of the 217 PHHI pancreases collected during surgery

in several European hospitals since 1976. This study was approved

by the ethical committee of the Universite Catholique de Louvain.

Whenever possible and after obtaining the written informed

consentoftheparents forthegenetic testingoftheirchildren,genetic

analyses were performed to search for ABCC8, KCNJ11, or GCK

genomic mutations. The genetic results of four of these 16 patients

have recently been published in a large series of 109 patients (30);

this previous publication evaluated the incidence and spectrum of

ABCC8 and KCNJ11 mutations in this cohort of PHHI.

Diagnoses of PHHI were made according to the clinical cri-

teria previously reported (8). The infants were considered to be

or to become nonresponsive to diazoxide therapy when 10 mg/

kg z d was insufficient to avoid hypoglycemic events (8).

Preoperative radiological evaluation was performed by pan-

creatic venous sampling (PVS) (10) in 14 cases, by 18F-PET (12,

13, 31) in one case, and with both in the last case.

All samples of partial pancreatectomies were fixed in 4% for-

malin to study SUR1, Ki67, and p57 and to perform molecular

biology investigations, including in situhybridization (ISH) forpro-

insulin mRNA and/or in Bouin’s solution for conventional micros-

copy on hematoxylin and eosin (H&E)-stained sections and the

immunostainingof insulin,proinsulin, somatostatin,andglucagon.

The anomalies were sometimes very small, being limited to

one or a few pancreatic lobules and were not detectable with the

naked eye. Moreover, the areas of interest were sometimes fixed

only in Bouin’s solution. All of the analyses were thus not per-

formed in all of the cases but were performed according to theadequacy of the available material.

The sizes of the b-cell nuclei and the b-cell nuclear crowding

(number of b-cell nuclei per 1000 mm2 of b-cell cytoplasm) were

2 Sempoux et al. Mosaicism in Infantile Hyperinsulinism J Clin Endocrinol Metab, December 2011, 96(12):0000–0000

Page 110: Développement du pancréas humain embryonnaire ex vivo / in vivo

measured in both types of islet (at least 10 islets in each case)according to themethod previously reported (6)Theareaof theseislets was measured and used to evaluate the differences betweenthe types of islet (Axiovision, Zeiss, Germany).

Immunoperoxidase staining was performed to detect insulin,proinsulin, and somatostatin as well as SUR-1, Ki67, and p57.The source of the antibodies and their dilution for use are givenin Supplemental Table 1 (published on The Endocrine Society’sJournals Online web site at http://jcem.endojournals.org).

Quantifications of Golgi proinsulin (diaminobenzidine) andsomatostatin cells (Fast Red) were performed on paraffin sec-tions. The total immunostained area of each islet was measured,and the coordinates of each islet were stored by an image ana-lyzer (KS-400 system; Zeiss-Vision, Munich, Germany). On thesame slides, insulin was further detected (Fast red), and the totalimmunostained area was measured again. After removal of theFast red staining by washing in alcohol, the Golgi proinsulin areawas measured, and the areas of somatostatin (d-cell area) andinsulin (b-cell area) immunolabeling were calculated. Then, theGolgi proinsulin-to-cytoplasmic insulin area ratio that reflectsthe b-cell synthesis function was calculated as previously re-ported (18) and related to the d-cell-to-b-cell area ratio. Isletsections (n 5 220) from six age-matched normoglycemic con-trols, born to nondiabetic mothers and dead from a disease not

related to the endocrine pancreas, were compared with 300 isletsfrom six infants with islet mosaicism.

The insulincontentofb-cells inboth typesof isletwasquantifiedon paraffin sections by measuring the specific absorbance (OD) on10 islets of each type after immunostaining for insulin according toa method of immunodensitometry previously reported (32).

ISH to detect proinsulin mRNA was performed with a flu-orescein isothiocyanate-labeled probe (final dilution 1:2;NCL-Proins, Novocastra, UK).

For microsatellite analysis, normal and hyperfunctional isletswere microdissected from paraffin sections of three cases (cases 8,12,and15;Table1)usingaPALMMicroLaserSystemspulsed laser(337 nm; Bernied, Germany) coupled to an Axiovert 200 micro-scope (Zeiss, Oberkochen, Germany). Although the morphologywas maintained using formalin, every sixth section from the con-secutive sections was stained with H&E to confirm the identifica-tion of normal and hyperfunctional islet areas. A Picopure DNAextraction kit (Arcturus Bioscience, Mountain View, CA) was usedto extract DNA from microdissected tissues following the manu-facturer’s recommendations. Loss of heterozygosity (LOH) wasstudied with a selection of markers located in the 11p15 region,from telomere to centromere (NCBI database): D11S4046 (0.8Mb), D11S2351 (3.9 Mb), D11S902 (16.3 Mb), and D11S4114(19.5 Mb), as described previously (18). PCR products were ana-

TABLE 1. Clinical data

Case Sex

Birthweight

(percentile)

Age atpresentation

(months)

Preop Dzxtreatment(mg/kg z d)

Localization(PVS or 18F-PET) Surgery Follow-up

1 F 46 3 6.5 Inconclusive (PVS) Partial tail Recurrence controlledby low-dose Dzx

2 F 78 6 10 Focal-corpus (PVS) Partial corpusand tail

Cured

3 M 94 8 10 Focal (PVS) Partial corpusand tail

Cured

4 F 4 5 12 Diffuse (PVS) Partial corpusand tail

Cured

5 F 64 6 15 Focal-tail (PVS) Partial tail Cured6 F 69 3 Sensitive

to DzxFocal (PVS) Partial corpus

and tailCured

7 M 79 4 Transientsensitivity

Tail (PVS) Tail Cured

8 M 48 5 15 Focal-head (PVS) Partial Recurrence controlledby low-dose Dzx

9 M 7 9 10 Focal tail (PVS) Partial tail Cured10 M 40 5.5 12 Focal tail (PVS) Partial tail Recurrence controlled

by low-dose Dzx11 F 51 6 6 Focal tail (PVS) Partial corpus

and tailCured

12 F 86 NN Transientsensitivity

Focal corpus (PVS) Partial corpusand tail

Cured

13 M 48 4 18 Focal corpus and tail(PVS)

Partial corpusand tail

Cured

14 F 90 NN 15 Inconclusive (PVS) Head corpus andtail

Recurrence controlledby low-dose Dzx

15 F Unknown 6 Sensitiveto Dzx

Inconclusive(18F-PET)

Partial head andisthmus

Recurrence controlledby low-dose Dzx

16 M 45 7 Transientsensitivity

Inconclusive(PVS and 18F-PET)

Corpus and tail Recurrence controlledby low-dose Dzx

Cases 4, 6, and 13 were not tested for ABCC8, KCNJ11, and GCK mutations. Mutations in the GDH gene have been ruled out in patients 7, 10,

and 14. Dzx, Diazoxide; F, female; M, male; Preop, preoperative.

J Clin Endocrinol Metab, December 2011, 96(12):0000–0000 jcem.endojournals.org 3

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lyzed using ABI Genescan Software. Allelic loss was scored if thearea under the allelic peak was reduced by a minimum of 50%. Apreviously tested focal lesion served as the positive control (casenumber 7 from Ref. 18).

The statistical evaluation of the results was conducted usingthe Fisher’s exact test for the differences in proportions of infantswith high birth weight and by Wilcoxon signed-rank test for allof the other parameters.

Results

Clinical data

The clinical data of the 16 atypical cases retrieved from

the patient files are detailed in Table 1. In most of the cases

(14 of 16), the hypoglycemic events occurred later in life

(median 165 d, range 1–270 d) than those in the classical

focal and diffuse forms, which are almost always diag-

nosed in the neonatal period (8, 9). The birth weights

expressed in percentiles were lower than in the forms pre-

viously reported (z score 5 10.04 vs. 11.36). Only one

patient (6%) had a percentile higher than 90% vs. 46% in

a series of 52 classical PHHI cases reported by de Lonlay-

Debeney et al. (8); this proportion of infants was signifi-

cantly lower than that previously reported (8) (P , 0.001,

Fisher’s exact test). At the onset of disease, all of the infants

were responsive to diazoxide at least for a short period of

treatment. Later, the sensitivity to the drug decreased, and

the infants required progressive and continuous increasing

of the dose, which, in certain infants, finally exceeded the

10 mg/kg z d that is characterized for diazoxide sensitivity

(8). Although they experienced marked improvement

upon diazoxide treatment, most infants remained prone to

occasional hypoglycemic events. This is strikingly dif-

ferent from the classical focal and diffuse forms that are

related to KATP channelopathies, which are almost al-

ways insensitive to diazoxide from birth (8, 9). Insulin

levels after PVS or 18F-PET were suggestive of focal

lesions in 11 cases, diffuse lesions in one case, and in-

conclusive or difficult to interpret in

four other cases.

For one or several reasons (persis-

tence of hypoglycemic events, progres-

sive loss of diazoxide sensitivity, or ma-

jor hypertrichosis and /or because PVS

was suggestive of a localized form), the

patients finally underwent surgery. Af-

ter partial pancreatectomy, 10 were de-

finitively cured. In the six others, hypo-

glycemic events reoccurred in the first

days or weeks after surgery and were

medically treated (Table 1).

Morphology

Conventional microscopy

Macroscopically, the pancreas ap-

peared normal, and no lesions were

identified macroscopically by the sur-

geon during surgery. Several biopsies

(from four to 20) were thus taken by the

surgeon in the area suspected by PVS or18F-PET or in different areas of the

gland when the preoperative localiza-

tion techniques were inconclusive, and

frozen sections were made. After recog-

nition of the abnormal area, biopsies

were taken to ensure that the limits of the

resection were healthy. The typical char-

acteristic of the lesions, already recogniz-

able on frozen sections, were confirmed

after H&E staining; these typical charac-

teristicswere thecoexistenceof twostrik-

FIG. 1. Islet mosaicism. A and B, On H&E-stained sections, the typical feature of this new

form of PHHI was the presence of both large hyperplastic islets with cytoplasm-rich b-cells

exhibiting some enlarged nuclei (A) as well as small shrunken islets that are 2-fold smaller (B);

C and D, after the immunodetection of insulin, the large islets showed a faint diffuse

homogeneous staining (C), whereas the small islets had strong polarized labeling (D); E, the

staining for proinsulin was stronger in large islets, suggesting a high level of function; F, this

staining was limited to a small dot in the small shrunken islets.

4 Sempoux et al. Mosaicism in Infantile Hyperinsulinism J Clin Endocrinol Metab, December 2011, 96(12):0000–0000

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inglydifferent typesof islet, resulting inamosaicpattern(Fig.

1, A–F).

Morphometry

Profiles from islets of the first type were 2.06-fold larger

than the second type of islet, with a mean value of 11,407

mm2 (P , 0.01). They contained numerous b-cells with

abundant cytoplasm and sometime large nuclei. However,

these nuclei were rarely as large as those in the classical

diffuse form of the disease (Fig. 1A). The radius of the

b-cell nuclei and the b-cell crowding have been evaluated

on both types of islet (Fig. 2) The b-cell nuclear mean

radius was constantly larger in the hyperplastic type 1

islets than in the small type 2 islets (5.66 6 0.83 vs. 3.85 6

0.21 mm, P , 0.01) (Fig. 2A). The b-cell nuclear crowding

was higher in small type 2 than in hyperplastic type 1 islets

(13.91 6 3.27 vs. 8.88 6 2.56) (Fig. 2B). The hyperplastic

type 1 islets were not observed throughout the whole pan-

creas but were confined to one or some adjacent lobules of

the pancreas (Fig. 3). Certain lobules contained only this

type of islet, but other lobules showed both types of islet.

The islets of the second type were small and appeared

hypotrophic. They contained b-cells with scanty cyto-

plasm and small nuclei and appeared suppressed (Fig. 1B).

This second type of islet was distributed throughout the

whole pancreas.

Immunocytochemistry, ISH, and microsatellite analysis

Immunocytochemistry also revealed striking differ-

ences between both types of islet (Fig. 1, C–F). In the first

type (large islets), the outlines of the islets labeled with

insulin antibody were very regular, and the cytoplasm of

the b-cells was only faintly stained at a very uniform level

(Fig. 1C), whereas the proinsulin antibody gave a strong

labeling, which marked a large Golgi area (Fig. 1E). The

insulin mRNA content, which was demonstrated by ISH,

was also abundant, and only a few somatostatin and glu-

cagon cells were present at the periphery of these islets

(Fig. 4A). In the second type of islet (small shrunken islets),

the staining with the antiinsulin antibody was strong, and

the outlines of the islets were irregular (Fig. 1D), but the

proinsulin antibody labeled only a small dot correspond-

ing to the Golgi area (Fig. 1F). Insulin mRNA labeling was

slightly lower in the islets, and the somatostatin and glu-

cagon cells were much more numerous than in the first

type of islet (Fig. 4B).

p57waspresent inbothtypesof islet (Fig.4,CandD),and

SUR1 immunolabeling had a similar pattern and intensity of

staining in both types of islet, appearing cytoplasmic with a

perimembranous reinforcement (Fig. 4, E and D).

The intensity of the insulin immunolabeling was mea-

sured in both types of islet by immunodensitometry. The

ODof insulin labelingwasalwayshigher in small suppressed

islets than in hyperplastic-hyperactive islets (0.403 1 0.101

vs. 0.288 1 0.093, P , 0.01) (Fig. 2C).

The proinsulin in the Golgi of b-cells, a reflection of

b-cell proinsulin synthesis, was measured in the islets from

normoglycemic patients (n 5 6) and from six cases with

FIG. 2. Nuclear radius, nuclear crowding, and densitometry of insulin.

The two symbols linked by a line represent the mean value of the

parameter measured in large hyperplastic islets (left) and in small

shrunken islets (right) in the same patient. A, Mean radius of 50

selected b-cell nuclei (micrometers) in large hyperplastic and small

shrunken islets; B, b-cell nuclear crowding (number of b-cell nuclei/

1000 mm2 of b-cell cytoplasm); C, specific absorbance (OD) of insulin

labeling (immunodensitometry); means of 10 islets in each islet type

and patients.

FIG. 3. Lobular distribution of large hyperplastic islets. At low

magnification, the lobular topography of hyperplastic-hyperactive islets

was already recognizable (proinsulin immunodetection).

J Clin Endocrinol Metab, December 2011, 96(12):0000–0000 jcem.endojournals.org 5

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islet mosaicism, and these measurements were compared

with the amount of d-cells in these islets. For this purpose,

the d-cell-to-b-cell area ratio (area percentage) was mea-

sured in each individual islet and related to the proinsulin

in the Golgi area (expressed as proinsulin-to-b-cell area

percentage) in the same islets. In the controls (Supplemen-

tal Fig. 1A), the d-cell-to-b-cell area ratio varied from

2.4 –119.0%. The Golgi proinsulin-to-insulin area ratio

showed weak variations (0–3.3%). By contrast, in hypo-

glycemic infants with islet mosaicism (Supplemental Fig.

1B), the d-cell-to-b-cell area ratio markedly varied within

the islets (from 0–250%) as did their Golgi proinsulin to

insulin area (from 0–21.6%). In this series of infants,

many islets with low d-cell-to-b-cell area ratios had large

Golgi proinsulin areas, whereas other infants with high

d-cell-to-b-cell area ratios had normal or small Golgi pro-

insulin areas (the small and shrunken islets).

The proliferation index of b-cells was counted in

both types of islet and was found to range 1.12–1.18%

of b-cells in suppressed and hyperplastic islets (not

significant).

Because the purity of the cell population was critical in

LOH detections, both types of islet were microdissected

from the same histological sections, and microsatellite

analysis was performed separately in the three cases (cases

8, 12, and 15). None of these showed an 11p15 LOH

involving imprinted genes in 11p15.5 and ABCC8, IGF2

or H-19, and KCNJ11 (Fig. 5). In patient 8, one marker

was not informative (D11S902); in patient 12, the four

markers were informative; and in patient 15, one marker

was not informative (D11S4114). To confirm that the la-

ser microdissection did not reduce the sensitivity for the

detection of LOH in small microdissected tissues by mic-

rosatellite analysis, a similar procedure was applied in a

case of focal adenomatous hyperplasia, and this procedure

confirmed the 11p15 deletion that was previously dem-

onstrated (case number 7 from Ref. 18) (Fig. 5).

FIG. 4. Insulin mRNA and somatostatin cells, p57, and SUR1. A and B,

Insulin mRNA and somatostatin cells in large hyperplastic islets (A) and

in small shrunken islets (B). Insulin mRNA labeling was darker in the

large hyperplastic islets, whereas d-cells were less numerous

(ISH/immunohistochemistry, differential interference contrast of

Nomarski). C and D, p57 immunodetection in large hyperplastic islets

(C) and in small shrunken islets (D). There were no differences in the

numbers of positive cells or in the intensities of the labeling. E and F,

SUR1 immunodetection showed no difference between the large

hyperplastic islets (E) and the small shrunken islets (F).

FIG. 5. Microsatellite analysis. A, Markers used and their distribution

in the 11p15 region; B and C, tracing corresponding to marker

D11S2351. In contrast to a classical KATP-related focal lesion used as

positive control (B), no LOH was observed in patients with histological

mosaicism (C, case 8). TM, Telomere; CM, centromere.

6 Sempoux et al. Mosaicism in Infantile Hyperinsulinism J Clin Endocrinol Metab, December 2011, 96(12):0000–0000

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Discussion

From our careful review of the series of 217 PHHI pa-

tients, we identified 16 cases (7.4%) with a unique pan-

creatic histology characterized by the coexistence of two

different types of islet, a striking mosaic aspect never de-

scribed in normoglycemic infants or in infants suffering

from the diffuse form of PHHI related to ABCC8 or

KCNJ11 mutations; unlike those with the mosaicism,

these pancreases have islets that have the same appear-

ance, with numerous small poorly formed islets and large

b-cell nuclei and cytoplasm (3, 5, 6). The novel mosaic

pattern differs from that of the focal form because of the

absence of adenomatous hyperplasia, but the islets of the

second type (with small nuclei and reduced cytoplasm)

resemble the normal resting islets located outside the focus

of insulin hypersecretion (3, 5, 6, 7, 18).

In the mosaic forms that we analyzed, the hyperactive

islets were located in one or some adjacent lobules. This

focal distribution explains why many cases were cured by

partial pancreatectomy.

The distinct aspects of the two types of islet suggest

major functional differences. The islets of the first type

contain b-cells with large nuclei and cytoplasm, charac-

teristic of hyperactive b-cells. Their proinsulin antibody-

labeled Golgi were large, reflecting a high rate of hormone

synthesis, as confirmed by a stronger labeling for insulin

mRNA by ISH. By contrast, the faint labeling of the an-

tiinsulin antibody suggested that insulin is not stored for

very long but is rapidly released. The islets of the second

type showed the opposite characteristics: small nuclei,

shrunken cytoplasm, and proinsulin-stained Golgi area

restricted to a punctate labeling but strong antiinsulin an-

tibody staining, indicating that these b-cells are resting

with a low production of proinsulin but a high storage of

insulin. This morphological aspect suggested that persis-

tent hypoglycemia inhibits insulin secretion and synthesis

in these b-cells, which implies that they remain normally

regulated.

In most cases, hyperactive islets contain only a few

d-cells compared with resting islets. Our quantitative anal-

ysis of the mosaic cases indicated that proinsulin synthesis

is higher in b-cells from islets with a low d-cell-to-b-cell

area ratio. The absence of d-cells may have a role in in-

fantile hyperinsulinism (33) but has never been demon-

strated in the islets of KATP hyperinsulinemic infants. The

meaningof the decrease in d-cell number in the hyperactive

islets of mosaic cases remains unclear. Suggested previ-

ously but not yet demonstrated (34), intra-insular soma-

tostatin secretion may play a role in the control of insulin

secretion. Furthermore, because the low d-to-b ratio also

observed in certain islets from normoglycemic controls

was not linked to proinsulin overproduction in these

controls, it is unlikely that the hyperproduction of pro-

insulin observed in hyperactive islets is caused by a rel-

ative lack of d-cells.

A lack of expression of the antiproliferative factor p57

has been implicated in the hyperplasia of classic focal le-

sions (17). Obvious expression of p57 in hyperfunctional

as well as resting islets of the mosaic form clearly indicates

that 11p15 LOH is not the cause of the lesion. This ob-

servation was confirmed in three cases using microsatellite

analysis. This new mosaic form of PHHI is thus distinct

and unrelated to the focal form previously described.

Large nuclei in the hyperactive islets should not be mis-

taken for a diffuse pathology (which would lead to ag-

gressive management) because they are restricted to a few

lobules and are mixed with small resting islets, a feature

never observed in the diffuse form of PHHI.

Interestingly, the clinical characteristics of these pa-

tients are also different from those of diffuse or focal PHHI

patients. Birth weight, corrected for gestational age and

sex, was indeed lower than that of infants suffering from

diffuse or focal KATP channelopathy (8, 9). This could

suggest that the disease was not present before birth or did

not cause major hyperinsulinism in utero. The observation

that the first symptoms of hypoglycemia occurred or were

recognized only later in life in most cases reinforced this

interpretation. This late onset may also result from the

small size of the lesion or from transient compensation by

physiological adaptive mechanisms.

The patients with mosaic histology shared some clinical

characteristics with infants presenting a loss-of-function

mutation of the HADH gene: a later onset in comparison

with KATP-deficient cases and a certain sensitivity to di-

azoxide. Although we cannot definitely rule out a role for

the HADH gene for now, no particular protein sensitivity

has been found in our mosaic cases, and the decrease in

diazoxide sensitivity with time that we observed has not

been reported in HADH-mutated patients.

In this series, 10 infants were definitively cured by a

partial pancreatectomy, but six were not cured. This could

suggest that their lesion was not localized or less localized

than expected and incompletely resected. In these six

cases, the observation of pancreatic lobules that contain

only healthy islets means that the lesion sparing at least

some lobules is thus a true localized lesion and does not

correspond to a classical diffuse form. It is likely that the

extension of the lesion, and then the difficulty of its re-

section, varies according to the chronology of the somatic

mutation occurrence.

When genetic analyses were performed, genomic

ABCC8 or KCNJ11 mutations were not found, indicating

that this mosaic form was also genetically different from

J Clin Endocrinol Metab, December 2011, 96(12):0000–0000 jcem.endojournals.org 7

Page 115: Développement du pancréas humain embryonnaire ex vivo / in vivo

those related to KATP channel abnormalities. This is in

agreement with the persistence of a response (although

incomplete) to diazoxide in these cases as well as with the

normal and similar SUR1 immunohistochemical expres-

sion we found in both types of islet. The observation of this

insular mosaicism, together with the lobular distribution

of the abnormalities, could be explained by the occurrence

of specific somatic mutations during the embryonic de-

velopment of the endocrine pancreas. These mutations

have so far not been identified in this series.

Rare cases of PHHI with histology that did not fit with

the classical description of focal or diffuse forms have al-

ready been reported (35, 36). Whether the particular his-

tological features could result from a longer evolution of

the KATP channel deficiency in children were operated on

later in life is unlikely because the usual KATP-deficient

children who are operated on later in life still present nu-

merous abnormal nuclei disseminated throughout the

pancreas and never show a mosaic pattern. The relative

sensitivity to diazoxide and the absence of demonstrated

genomic ABCC8 or KCNJ11 mutations in these patients

also do not favor this hypothesis.

Pancreatic b-cell nuclear enlargement that is found only

in some sections of the pancreas has been recently reported

in a hypoglycemic infant by Hussain et al. (37). That case,

however, completely differs from ours by its mosaic uni-

parental paternal disomy, with the interstitial region of the

uniparental paternal disomy encompassing the KATP

channel genes and including an ABCC8 mutation, which

was not found in the 13 patients we analyzed for genetic

mutations. Furthermore, the patient had a neonatal onset

of hypoglycemia and an immediate and total insensitivity

to diazoxide.

We have identified a new form of PHHI that is char-

acterized by a morphological mosaicism, with particular

histological, immunohistochemical, and clinical charac-

teristics. The abnormal hyperfunctional islets of this form

are most often confined to a few adjacent lobules. The

genetic background of these cases remains unclear, but the

diagnosis of morphological mosaicism should be sus-

pected from the peculiar clinical presentation and can be

confirmed during surgery using intraoperative surgical

frozen sections, with the knowledge that partial pancre-

atectomy may often be sufficient to cure the infant.

Acknowledgments

The authors thank Mrs. J. Marchandise, Mr. S. Godecharles, andMr. S. Lagasse for their skillful technical assistance.

Address all correspondence and requests for reprints to: Prof.Dr. Jacques Rahier, M.D., Ph.D., Department of Pathology, Cli-

niques Universitaires Saint Luc, Avenue Hippocrate 10, 1200Brussels, Belgium. E-mail: [email protected].

This work was supported by Grants 3.4616.05 (to J.R.),9.4559.04 (to C.S.), and 1.5.216.04 (to C.S.) from the FondsNational de la Recherche Scientifique Medicale (Belgium) and byARC Grant 05/10-328 (to C.S.) from the General Direction ofScientific Research of the French Community of Belgium.

Disclosure Summary: The authors have nothing to disclose.

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The surgical management of atypical forms of congenital

hyperinsulinism

Carmen Capito, MD,a Pascale de Lonlay, MD, PhD,b Virginie Verkarre, MD,c

Francis Jaubert, MD,c Jacques Rahier, MD,d Claire Nihoul-Fékété, MD,a

Yves Aigrain, MDa

From the aDepartment of Pediatric Surgery, AP-HP Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris,

France;bReference Center of Metabolic Diseases, AP-HP Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris,

France;cDepartment of Pathology, AP-HP Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France; and thedDepartment of Pathology, Cliniques Universitaires, St-Luc, Louivain University, Brussels, Belgium

Beyond the 2 classical forms of congenital hyperinsulinism, focal and diffuse, we report our experience

on the surgical treatment of atypical forms. We define 2 subtypes among these atypical forms of

hyperinsulinism: in case of a giant focal form the surgical strategy is the same as in focal forms. In case

of hyperinsulinism caused by a mosaic, our experience suggests the benefit of a limited resection from

the tail to the body of the pancreas.

© 2011 Elsevier Inc. All rights reserved.

KEYWORDSHyperinsulinism;

Atypical forms;

Surgery

Therapeutic management of congenital hyperInsulinism

(CHI) may be challenging.1,2 Some patients (43% in our

experience) will require surgical treatment because of the

inability to medically control the disease and avoid delete-

rious hypoglycemic episodes.2-4 Histologic classification of

CHI includes 2 major forms: diffuse forms, which are char-

acterized by hyperfunction of every islet of Langerhans, and

focal forms, which correspond to an adenomatous hyper-

plasia and are smaller than 15 mm with resting islets outside

the lesion.2,5-7 Diffuse forms, which are uncontrollable by

medical treatment, will require a near total pancreatectomy,

with unpredictable postoperative results.2 Focal forms, as

we proposed a few years ago,8 can be cured by complete

resection of the lesion with pancreatic-sparing surgery when

possible (efficient peroperative pathologic examination on

frozen sections for free margins). A third group correspond-

ing to rare case reports in the literature9 includes what is

generally called atypical forms, which are defined as non-

focal and nondiffuse forms on histologic reports. These

atypical forms are not homogeneous, and we have been able

to identify 2 varieties: segmental mosaic forms and exten-

sive focal forms.

Materials and methods

From 1991 to 2009, 427 children were managed for CHI in

our center. They all presented with hypoglycemia less than

3 mmol/L associated with inappropriate plasma insulin lev-

els. Among them, 184 patients were operated on for CHI

resistant to medical treatment. Before surgery, a radiologi-

cal evaluation was performed by pancreatic venous cathe-

Address reprint requests and correspondence: Yves Aigrain, MD,

Service de Chirurgie Pédiatrique, 149 Hôpital Necker Enfants-Malades rue

de Sèvres, 75743 Paris Cedex 15 France.

E-mail: [email protected].

1055-8586/$ -see front matter © 2011 Elsevier Inc. All rights reserved.

doi:10.1053/j.sempedsurg.2010.10.003

Seminars in Pediatric Surgery (2011) 20, 54-55

Page 118: Développement du pancréas humain embryonnaire ex vivo / in vivo

terization sampling10 or, after 2002, 18-fluoro-L-Dopa

positron-emission tomography scanning.11 These examina-

tions permitted us to diagnose the histologic classical form

of CHI and, in case of focal forms, to locate the lesion and

guide the surgical procedure.12 The indications for surgery

were assumed in every case of focal CHI, and a partial

elective pancreatectomy was performed.8,13 Diffuse lesions

were operated whenever complete suppression of hypogly-

cemia was not achieved by a medical treatment compatible

with home management. A near-total pancreatectomy was

performed, leaving only the pancreatic tissue in the concav-

ity of the duodenum and containing the choledocal duct.13

The operative protocol consisted in all cases of an inspec-

tion of the anterior wall of the gland searching for an

obvious lesion. Three biopsies were taken in the 3 morpho-

logic areas of the pancreas for pathologic confirmation of

histologic form of the disease on frozen sections. Focal form

is defined as an adenomatous hyperplasia smaller than 15

mm and made by endocrine cells with an abundant cyto-

plasm and large irregular nuclei in the hyperinsulinic zone

and resting b cells with little nuclei outside the focal lesion.

Diffuse forms are defined as hyper function of every islet of

Langerhans within the pancreas. Those islets include abnor-

mal beta cells with giant nuclei. Atypical forms were de-

scribed as nonfocal and nondiffuse form. After surgery,

specimens were examined from a confirmation of the his-

tologic form and an adequate margin of normal tissue. We

will focus on the surgical treatment of patients whose patho-

logic report was atypical because lesions encountered were

not in accordance with the classical definition of focal or

diffuse forms.

Results and conclusions

Histopathological results

The review of the pathologic reports allows separating the

patients into 2 groups. The first group has pathologic fea-

tures that were similar to classical focal forms but the size

of the lesion was much larger than 15 mm as classically

described in typical focal forms. We called this pathologic

entity “extensive focal form.” In the second group the pan-

creatic architecture was normal but the coexistence of hyper

functional islets with some abnormal giant beta cell nuclei,

and resting small and round islets (mosaicism), in a seg-

mental distribution. Because of this segmental distribution

and the mixed lesion with parts of diffuse (hyper functional

islets) and focal lesion (resting islets) we called this form

“segmental mosaic form.”

Extensive focal forms

In this group the surgical procedure consists in the resection

of the focal lesion. The main difference with the classic

focal forms concern the number of biopsies needed to reach

free margins, the length of surgery and the amount of

pancreatic tissue removed. Moreover, a redo procedure with

further resection due to persistent hypoglycemia is more

frequent in this group of patients.

Segmental mosaic forms

Anatomical location of the lesion involved in most cases the

tail and body of the pancreas. That is why we strongly

advocate a partial resection starting from the left to the

portal vein. Even in cases where this limited procedure does

not completely cure the hypoglycemia, most patients are

improved by the limited resection and few will require a

redo procedure.

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55Capito et al Surgical Management of Atypical Forms of CIH